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Gluconic acid end-groups in unbleached cotton cellulose
144
CARBOHYDRATERESEARCH
Note
Gluconic
acid end-groups
KENNETH LARSON Department
of
AND
OLOF
Engineering
in unbleached cotton
cellulose
SAMUELSON
Chemistry,
Chalmers
Tekniska
Htigskola,
GLiteborg
(Szceden)
(Received February 12th, 1969)
Great difficulties are encountered in the determination cf the structure of the end groups in unbleached cchulose. Firstly, the number of end groups is very small, and secondly, appreciable proportions of polysaccharides other than cellulose are present in the unbleached cotton’. These polysaccharides contain uranic acid units w.tich interfere with the determination of acidic end-groups present in the cellulose. In most cotton samples, the number of carboxyl groups is larger than the number of end groups calculated from molecular weight determinations, even in samples subjected to purification by extraction with hot alkali. No conclusions about the presence of carboxyl end-groups in undegraded cellulose can therefore be drawn from experiments of this type. A three-charnel analyzer coupled with chromatography on anion-exchange resins makes it possible to detect small amounts of aldonic acids in the presence of large amounts of uranic acids2. In one channel, all oxidizable acids are determined by chromic acid oxidation. In a second channel, the eluate is treated with carbazole which gives a strong reaction with uranic, but no reaction with aldonic, acids. The third channel is employed for periodate oxidation with subsequent determination of formaldehyde. Aldonic acids give a strong response, whereas uranic acids give no, or a very slight, response. A chromatographic study of the non-volatile, monoprotic acids isolated from a hydrolyzate of unbleached cotton revealed that a variety of acids were present. A chromatogram from a run in O.OShfsodium acetate is reproduced in Fig. 1. As expected, a Iarge amount of Ievulinic acid (L) was recorded. Another artefact is an anhydrosaccharinic acid (AS) found to be present in all hydrolyzates from cellulose prepared by the applied method 3. Galacturonic acid (Ga) was the preponderant uranic acid, which confirms that raw cotton contains appreciable proportions of pectic substances’. As expected from previous work4.‘, 2-U-(4-O-methyl-a-D-glucopyranosyluronic acid)-D-xylose (MGX) and 4U-methylglucuronic acid (MG) were present in large amounts. The chromatograms confh-m, moreover, the earlier observation that cellobiouronic (C) and glucuronic (Gu) acids were present5. Another biouronic acid, with properties similar to those of 2-0-(4-O-methyl-a-D-glucopyranosyluronic acid)-D-xylose, was indicated as well. No attempts were made to identify this acid. C’cz~6ohycf. Res.,
II
(1969)
144-147
NOTE
145
Chart reading, mm a MGX
101
L-
_ _.__S’_-2‘.-_
-_d--’
“OL
100
. --__----_----------Eluate
volume,m!
Fig. I. Separation of monoprotic acids isolated from 1.5 g of solvent-extracted Peruvian cotton. Column: Dowex l-x8; 23-24 ,~m: 1150 x 3.8 mm. Eluent: 0.08~ sodium acetate with acetic acid added to obtain pH 5.9; , chromic acid method; - - - -, carbazole method; -=-e---.-,
periodate-formaldehyde
method.
A distinct peak with the position of gluconic acid (G) was recorded in the channel. In 0.5~ acetic acid, gluconic acid appears at a retention volume very close to that of 2-O-(4-O-methyl-cr-D-glucopyranosyluronic acid)-D-xylose5, and a run in this medium showed that a distinct peak was recorded with periodate at the relevant position. Gluconic acid was isolated on a preparative scale from 100 g of cotton by chromatography in 0.08~ sodium acetate, which gave a mixture containing mainly 4-O-methylglucuronic acid. This mixture was rechromatographed in 0.5M acetic acid. A cleancut separation was obtained’. The purity of the isolated gluconic acid was checked on the automatic column. The distribution coefficients and the response indices’ agreed with those recorded with an authentic sample. The 1,4iactone was trimethylsilylated and studied by gas chromatography-mass spectrometry 6. The results confirmed that the isolated acid consisted of gluconic acid. To study whether the gluconic acid was present as end groups in the cellulose or in some non-cellulosic impurity, the raw cotton was purified by boiling with alkali7. The chromatograms showed that galacturonic acid had almost completely disappeared, but that appreciable amounts of the other uranic acids were still present. Peaks corresponding to gluconic acid were recorded in runs both in 0.08~ sodium acetate and in 0.5~ acetic acid. Chromatography of the sugar fraction obtained after hydrolysis revealed that appreciable amounts of xylose were present’. The alkali-boiled cellulose was further purified by subjecting it to a mild treatment with acid and a subsequent extraction with 6% sodium hydroxide at room temperature_ The chromatograms indicated that gluconic acid was present, although in a smaller amount than in the non-hydrolyzed sample. Likewise, 40-
periodate
Curbuhyd. Res., 11 (1969) 144-147
NOTE
methylglucuronic acid and 2- 0-(4-O-methyl-cL-D-glucopyranosyluronic acid)-D-xylose were present in about the same amounts as gluconic acid. The other uranic acids were not present in detectable amounts. The sugar analysis showed that small amounts of xylose were still presents, and that the total amount of aldoses other than glucose was about 0.05%. The gluconic acid was isolated on a preparative scale and identified. To ascertain that gluconic acid was not an artefact formed by oxidation during the hydrolysis or during the procedure used for its isolation, blank experiments were made with glucose under identical conditions. No gluconic acid was detected in the acid fraction which contained large amounts of levulinic acid, minor amounts of the anhydrosaccharinic acid referred to above, and trace amounts of some unidentified acids. Blank tests with gluconic acid showed that the losses were less than 10% during the whole procedure. The results indicate that the applied method gives a reliable determination of gluconic acid groups in cellulose materials. The amount of gluconic acid in the hydrolyzate from the raw cotton (purified by solvent extraction only) was 16 mg/lOO g of cotton. The corresponding figure obtained with the alkali-boiled, hydrolyzed, and cold alkali-extracted cotton was 4 mg. As shown previously, gluconic acid end-groups present in large amounts in bleached~cellulose are split off fairly easily during a partial hydrolysis of the cellulose4. In addition, low-molecular fragments of the cellulose molecules are extracted together with the non-cellulosic material during the extraction with cold alkali. For these reasons, a lower figure was expected with the hydrolyzed and cold alkali-extracted sample. Unfortunately, the removal of non-cellulosic material from the cotton was unsatisfactory, unless the sample was subjected to an acid treatment before the tinal extraction with alkali. Since the hydrolyzed and alkali-extracted sample contained only traces of other polysaccharides, it can be concluded that the presence of gluconic acid in the hydrolyzate can be ascribed to gluconic acid end-groups in the cellulose chains. The experimental results do not permit a decision about whether all, or only a fraction, of the gluconic acid groups in the raw cotton originate from the terminal units in the cellulose chains. It is likely that other end groups can be present as well. For these reasons, it is risky to apply determinations of gluconic acid groups in cotton to the determination of the degree of polymerization. EXPERIMENTAL
The cotton samples were extracted with ethanol for 18 h and with dichloromethane for 18 h, and air dried at room temperature. One of the samples (Peruvian cotton) was not purified further. Another sample (American cotton) was purified by alkali cooking’, then subjected to acid hydrolysis in boiling 0.05~ sulfuric acid for 3 h, and finally extracted for 2 min at room temperature with 6% sodium hydroxide. The cellulose samples were dissolved in hydrochloric acid and, after hydrolysis, the non-volatile, monoprotic acids were isolated as a group according to a method Carbohyd. Res., 11 (1969) 144-147
147
NOTE
described previously’. Chromatographic separations of the organic acids were carried out both on a preparative scale (100 g of cotton) and on automatic coIumns2. The chromic acid oxidation and the periodate oxidation (0.015,~ sodium metaperiodate buffered at pH 7.0; 25”; residence time, 2 min) were carried out as described previously2. The length of the flow cell in the carbazole channel was increased to obtain a higher response
with uranic acids.-The
identifications
were made as described
in
the papers referred to above3- ‘. ACKNOWLEDGMENTS
The authors express their thanks to the Swedish Council for Applied Research for financial support. REFERENCES 1 2 3 4 5 6 7 8 9
G. v. HORNUFF AND H. RICHTER, Faserforsch. Te.rfifrech.. 15 (1964) 115. B. CARLSSON, T. TSAKSSON,AND 0. SAMUELSON, Anal. ChimAcra,43 (1968)47. S. PETTERSSON AND 0. SA~KJELSON,Svensk Papperstid., 72 (1969) 261. I. NOR~TEDT AND 0. SAMUELSON, Svensk Papperstid., 68 (1965) 565. S. PETTERSSON AND 0. SAMUELSON, Scensk Pappextid., 71 (1968) 429. G. PETERSSON,0. SAhmxsoN, K. ANJOU, AND E.v. SYDOW, Acfa ChemScand.,21 C. DOREE, Methods of Celhdose Chemistry, Chapman and Hall, London, 1950. L.-I. LARS~ON AND 0. SAMUELSON, Svensk Papperstid., 71 (1968) 432. 0. SAh%mLsoN AND L. THEDE, Tappi, 52 (1969) 99.
(1967) 1251.
Carbohyd. Res., 11 (1969) 144-147
CARBOHYDRATERESEARCH
Note
Gluconic
acid end-groups
KENNETH LARSON Department
of
AND
OLOF
Engineering
in unbleached cotton
cellulose
SAMUELSON
Chemistry,
Chalmers
Tekniska
Htigskola,
GLiteborg
(Szceden)
(Received February 12th, 1969)
Great difficulties are encountered in the determination cf the structure of the end groups in unbleached cchulose. Firstly, the number of end groups is very small, and secondly, appreciable proportions of polysaccharides other than cellulose are present in the unbleached cotton’. These polysaccharides contain uranic acid units w.tich interfere with the determination of acidic end-groups present in the cellulose. In most cotton samples, the number of carboxyl groups is larger than the number of end groups calculated from molecular weight determinations, even in samples subjected to purification by extraction with hot alkali. No conclusions about the presence of carboxyl end-groups in undegraded cellulose can therefore be drawn from experiments of this type. A three-charnel analyzer coupled with chromatography on anion-exchange resins makes it possible to detect small amounts of aldonic acids in the presence of large amounts of uranic acids2. In one channel, all oxidizable acids are determined by chromic acid oxidation. In a second channel, the eluate is treated with carbazole which gives a strong reaction with uranic, but no reaction with aldonic, acids. The third channel is employed for periodate oxidation with subsequent determination of formaldehyde. Aldonic acids give a strong response, whereas uranic acids give no, or a very slight, response. A chromatographic study of the non-volatile, monoprotic acids isolated from a hydrolyzate of unbleached cotton revealed that a variety of acids were present. A chromatogram from a run in O.OShfsodium acetate is reproduced in Fig. 1. As expected, a Iarge amount of Ievulinic acid (L) was recorded. Another artefact is an anhydrosaccharinic acid (AS) found to be present in all hydrolyzates from cellulose prepared by the applied method 3. Galacturonic acid (Ga) was the preponderant uranic acid, which confirms that raw cotton contains appreciable proportions of pectic substances’. As expected from previous work4.‘, 2-U-(4-O-methyl-a-D-glucopyranosyluronic acid)-D-xylose (MGX) and 4U-methylglucuronic acid (MG) were present in large amounts. The chromatograms confh-m, moreover, the earlier observation that cellobiouronic (C) and glucuronic (Gu) acids were present5. Another biouronic acid, with properties similar to those of 2-0-(4-O-methyl-a-D-glucopyranosyluronic acid)-D-xylose, was indicated as well. No attempts were made to identify this acid. C’cz~6ohycf. Res.,
II
(1969)
144-147
NOTE
145
Chart reading, mm a MGX
101
L-
_ _.__S’_-2‘.-_
-_d--’
“OL
100
. --__----_----------Eluate
volume,m!
Fig. I. Separation of monoprotic acids isolated from 1.5 g of solvent-extracted Peruvian cotton. Column: Dowex l-x8; 23-24 ,~m: 1150 x 3.8 mm. Eluent: 0.08~ sodium acetate with acetic acid added to obtain pH 5.9; , chromic acid method; - - - -, carbazole method; -=-e---.-,
periodate-formaldehyde
method.
A distinct peak with the position of gluconic acid (G) was recorded in the channel. In 0.5~ acetic acid, gluconic acid appears at a retention volume very close to that of 2-O-(4-O-methyl-cr-D-glucopyranosyluronic acid)-D-xylose5, and a run in this medium showed that a distinct peak was recorded with periodate at the relevant position. Gluconic acid was isolated on a preparative scale from 100 g of cotton by chromatography in 0.08~ sodium acetate, which gave a mixture containing mainly 4-O-methylglucuronic acid. This mixture was rechromatographed in 0.5M acetic acid. A cleancut separation was obtained’. The purity of the isolated gluconic acid was checked on the automatic column. The distribution coefficients and the response indices’ agreed with those recorded with an authentic sample. The 1,4iactone was trimethylsilylated and studied by gas chromatography-mass spectrometry 6. The results confirmed that the isolated acid consisted of gluconic acid. To study whether the gluconic acid was present as end groups in the cellulose or in some non-cellulosic impurity, the raw cotton was purified by boiling with alkali7. The chromatograms showed that galacturonic acid had almost completely disappeared, but that appreciable amounts of the other uranic acids were still present. Peaks corresponding to gluconic acid were recorded in runs both in 0.08~ sodium acetate and in 0.5~ acetic acid. Chromatography of the sugar fraction obtained after hydrolysis revealed that appreciable amounts of xylose were present’. The alkali-boiled cellulose was further purified by subjecting it to a mild treatment with acid and a subsequent extraction with 6% sodium hydroxide at room temperature_ The chromatograms indicated that gluconic acid was present, although in a smaller amount than in the non-hydrolyzed sample. Likewise, 40-
periodate
Curbuhyd. Res., 11 (1969) 144-147
NOTE
methylglucuronic acid and 2- 0-(4-O-methyl-cL-D-glucopyranosyluronic acid)-D-xylose were present in about the same amounts as gluconic acid. The other uranic acids were not present in detectable amounts. The sugar analysis showed that small amounts of xylose were still presents, and that the total amount of aldoses other than glucose was about 0.05%. The gluconic acid was isolated on a preparative scale and identified. To ascertain that gluconic acid was not an artefact formed by oxidation during the hydrolysis or during the procedure used for its isolation, blank experiments were made with glucose under identical conditions. No gluconic acid was detected in the acid fraction which contained large amounts of levulinic acid, minor amounts of the anhydrosaccharinic acid referred to above, and trace amounts of some unidentified acids. Blank tests with gluconic acid showed that the losses were less than 10% during the whole procedure. The results indicate that the applied method gives a reliable determination of gluconic acid groups in cellulose materials. The amount of gluconic acid in the hydrolyzate from the raw cotton (purified by solvent extraction only) was 16 mg/lOO g of cotton. The corresponding figure obtained with the alkali-boiled, hydrolyzed, and cold alkali-extracted cotton was 4 mg. As shown previously, gluconic acid end-groups present in large amounts in bleached~cellulose are split off fairly easily during a partial hydrolysis of the cellulose4. In addition, low-molecular fragments of the cellulose molecules are extracted together with the non-cellulosic material during the extraction with cold alkali. For these reasons, a lower figure was expected with the hydrolyzed and cold alkali-extracted sample. Unfortunately, the removal of non-cellulosic material from the cotton was unsatisfactory, unless the sample was subjected to an acid treatment before the tinal extraction with alkali. Since the hydrolyzed and alkali-extracted sample contained only traces of other polysaccharides, it can be concluded that the presence of gluconic acid in the hydrolyzate can be ascribed to gluconic acid end-groups in the cellulose chains. The experimental results do not permit a decision about whether all, or only a fraction, of the gluconic acid groups in the raw cotton originate from the terminal units in the cellulose chains. It is likely that other end groups can be present as well. For these reasons, it is risky to apply determinations of gluconic acid groups in cotton to the determination of the degree of polymerization. EXPERIMENTAL
The cotton samples were extracted with ethanol for 18 h and with dichloromethane for 18 h, and air dried at room temperature. One of the samples (Peruvian cotton) was not purified further. Another sample (American cotton) was purified by alkali cooking’, then subjected to acid hydrolysis in boiling 0.05~ sulfuric acid for 3 h, and finally extracted for 2 min at room temperature with 6% sodium hydroxide. The cellulose samples were dissolved in hydrochloric acid and, after hydrolysis, the non-volatile, monoprotic acids were isolated as a group according to a method Carbohyd. Res., 11 (1969) 144-147
147
NOTE
described previously’. Chromatographic separations of the organic acids were carried out both on a preparative scale (100 g of cotton) and on automatic coIumns2. The chromic acid oxidation and the periodate oxidation (0.015,~ sodium metaperiodate buffered at pH 7.0; 25”; residence time, 2 min) were carried out as described previously2. The length of the flow cell in the carbazole channel was increased to obtain a higher response
with uranic acids.-The
identifications
were made as described
in
the papers referred to above3- ‘. ACKNOWLEDGMENTS
The authors express their thanks to the Swedish Council for Applied Research for financial support. REFERENCES 1 2 3 4 5 6 7 8 9
G. v. HORNUFF AND H. RICHTER, Faserforsch. Te.rfifrech.. 15 (1964) 115. B. CARLSSON, T. TSAKSSON,AND 0. SAMUELSON, Anal. ChimAcra,43 (1968)47. S. PETTERSSON AND 0. SA~KJELSON,Svensk Papperstid., 72 (1969) 261. I. NOR~TEDT AND 0. SAMUELSON, Svensk Papperstid., 68 (1965) 565. S. PETTERSSON AND 0. SAMUELSON, Scensk Pappextid., 71 (1968) 429. G. PETERSSON,0. SAhmxsoN, K. ANJOU, AND E.v. SYDOW, Acfa ChemScand.,21 C. DOREE, Methods of Celhdose Chemistry, Chapman and Hall, London, 1950. L.-I. LARS~ON AND 0. SAMUELSON, Svensk Papperstid., 71 (1968) 432. 0. SAh%mLsoN AND L. THEDE, Tappi, 52 (1969) 99.
(1967) 1251.
Carbohyd. Res., 11 (1969) 144-147