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A method for the analysis of sugars in plant cell-wall polysaccharides by gas-liquid chromatography
340 A method
NOTES
for
polysaccharides
the
analysis
of sugars
by gas-liquid
in plant
cell-wall
chromatography
Numerous attempts have been made to determine the sugar composition of plant cell-wall polysaccharides 1--4 by laborious methods which are subject to large experimental errors. Apromising approach to the analysis of monosaccharides involves the conversion of the sugars into their respective alditol acetates, which are then separated by gas-liquidchromatographySy6. This method has been used for analyzing the acid hydrolyzates of polysaccharides 7v8, but, although it has several advantages over earlier methods of polysaccharide analysis, it is unsuitable for analysis of a large number of samples. Modifications that permit rapid and reproducible analysis of the sugar constituents of polysaccharides of plant cells are here described. Trifluoroacetic acid was used for hydrolyzing the polysaccharides, because (a> the yields of most monosaccharides from plant cell-wall preparations are at least equal to those obtained by hydrolysis with mineral acids, and (b) it may be readily removed by evaporation. Fig. 1 shows the yields of sugars obtained from cell walls of pinto-bean hypocotyls on hydrolysis with 2~ trifluoroacetic acid in a sealed tube at 121o for various periods of time. In general, 2~ trifluoroacetic acid (2 ml) was added to the wall material (20 mg); myo-inositol (1 mg) was added as an internal standard, as it is not present in cell walls and has a retention time quite different from those of the various sugars of
8r
TIME
(HOURS)
Fig. 1. Infiuence of the length of hydrolysis on the determination of the carbohydrate component8 of pinto-bean hypocotyl cell-walls. The cell walls were hydrolyzed with 2~ trifluoroacetic acid at 121 o for various periods of time.
Cprbohyd.Res., 5 (1967) 340-345
341
NOTES
the wall. ,4fter hydrolysis for 1 h (optimal for most of the sugars), the soluble portion was evaporated to dryness at 50” under a stream of Altered air. The sugar mixtures obtained from hydrolyzed polysaccharides were reduced to their respective alditols with sodium borohydride (10 mg) in N ammonia (OS ml). The ammonia was added to ensure that those solutions that contained uranic acids and/or traces of trifluoroacetic acid were alkaline during reduction. After 1 h at room temperature (found sufficient for complete reduction of aldoses), the excess of borohydride was decomposed by dropwise addition of glacial acetic acid, un&il effervescence had ceased_ The borate produced on decomposition of the borohydride forms a complex with the alditols, and retards their acetylation. It was found that removal of boric acid improved the acetylation. A mixture of aldoses (rhanmose, xyiose, and glucose) and alditols (arabinitol and mannitol) was subjected to the reduction. To aliquots (1 ml) of this mixture in test tubes, 1 to 8 additions of methanol (1 ml) were made, each followed by evaporation to dryness at 50” under a stream of filtered air. Acetic anhydride (1 ml) was then added and the tubes were sealed and heated for 1 h at 100”. It is evident from Fig. 2 that a stigle addition of methanol had little effect on the degree -8
-7
-6
%
-6
s 9
-4
n b LI
-3
ii! ii? %
t 2
kl_____. , , 0
1
2
3
Additions
4
5
of
methanol
6
1
s
Fig. 2. Influence of the number of additions of methanol on the degree of acetylation. Aliquots (1 ml) were transferred from a mixture (10 ml) containing 1 mg of L-rhamnose, L-arabinitol, r+xylose, D-mannitol, and D-glucose per ml into each of 9 test tubes. These samples were reduced with N ammonia (0.5 ml) containing sodium borohydride (10 mg) and then subjected to various numbers of sequential additions of 1 ml of methanol with evaporation to dryness after each addition. The number of additions of methanol differed from tube to tube. The samples were acetylated, and assayed. as described in the text.
Carbohyd. Rcs., 5 (1967) 340-345
342
NOTES
to which the alditols were acetylated (see below for details on chromatography). However, additional methanol evaporations, up to a total of 4, increased the degree of acetylation. Quantitative analysis of the boric acid remaining after each evaporation’ showed that 5 additions and evaporations of methanol gave optimal results. Acetylation of the alditols was performed in a sealed tube with acetic anhydride (1 ml) heated for 3 h at 121”. The sodium acetate remaining after the removal of boric acid (as its trimethyl ester) served as the basic catalyst for the acetylation reaction. Pyridine, used for this purpose by previous workers, had a deleterious effect on the column material and produced severe tailing of the peaks, especially of the solvent peak. The best g.1.c. separation was obtained with a liquid phase containing 0.2% of poly(ethylene glycol succinate), 0.2% of poly(ethylene glycol adipate) and 0.4% of silicone XFl150 coated on Gas-Chrom P (100-120 mesh) (Applied Science Labs., State College, Pa.). The dried material (ca. 1.1 g) was packed by vibration into a copper tube (4 ft x l/8 in O.D.). A balancing column (5 ft long), containing about 1.4g of the same material, was similarly packed. The columns were conditioned at 180” for 4 h. Separations with this column material were superior to those previously reported 6g8. Fig. 3 illustrates the response of a 5.0-mV recorder to a l-,~l injection of an acetic anhydride solution containing 1 pg of each of the following: the pentaacetates -SO-
-4.0Y >
Rha
-5 z
P
-3.o-
% k z E
-2.o-
,o 8 ii -l.O-
-
0 5 1 TEMPERATURE
(‘C.)
Fig. 3. Mustration of the recorder response for a l-_A injection of acetic anhydride containing 1 pg each of L-rhamnose, D-fucose, L-arabinose, D-xylose, D-mannose, D-galactose, D-glucose, and myo-inositol after reduction and acetylation. The F & M gas chromatograph electrometer \vas set at range 10 and attenuation 1. Full-scale response on the strip-chart recorder WE+5.0 mV. The injcction-port temperature was 210”, and the detector temperature was 250”. The column temperature on injection was 120’; this was maintained for 10 min after injection, and then raised by lo per min. Carbohyd. Res., 5 (1967)
340-345
NOTES
343
of arabinitol, fucitol, rhamnitol, and xylitol, and the hexaacetates of galactitol, glucitol, mannitol, and myo-inositol. The sensitivity achieved in this procedure (see Fig. 3) was obtained by setting the electrometer at range 10 and the attenuation at 1. The area under each peak was measured by electronic- integration. The output of the gas chromatographs was recorded on instrumentation-quality magnetic tape (Scotch Brand X 860, Minnesota Mining and Manufacturing Co.) with an Infotronics Model CRS-43RlD magnetic tape recorder, and was monitored by means of an Infotronics Model CRS-40TS playback system at 16 times the recording speed (7.5 in/set). Integration of the response peaks was achieved with an Infotronics digital readout system Model CRS-1 lHS/42. Results obtained in the analysis of cell-wall polysaccharides are given in Fig. 4. The cell walls analyzed in this experiment were obtained from sycamore (Acer pseudoplatanus) cells grown in suspension culture’o on a defined (MID) medium*. A sample of cell wall (16 mg) was hydrolyzed, and reduced, and then acetylated with acetic anhydride (1 ml). One 1.11was injected into the gas chromatograph to give the recorder responses shown in Fig. 4. The accuracy and precision of this method of analysis were tested as follows (Table I): samples, each containing individually weighed amounts of the 7 sugars -5.0
-
-4.o7 > .5 :
--3.o-
% % 2
Rha -*o-
5 2 8 z
- 1.0-
-o1 TEMPERATURE
(‘C.)
Fig- 4. Gas-chromatographic analysis ofa I+1 injection containing the material from sycamore (Acer pse&q&tanus) cell-walls. The cell walls were hydrolyzed, and the products reduced, acetylated, and analyzed as described in the text. *Personal communication from Philip Filner, Michigan State University. Carbohyd. Res., 5 (1967) 340-345
344
NOTES
found in plant cell-walls and myo-inositol (1 mg/ml), were subjected to acid hydrolysis, reduction, acetylation, and assay by gas chromatography. L-Arabinose, D-fucose, TABLE PRECISION
Injecrion
I AND
ACCURACY
OF THE
METHOD
Proportion of sugar components after repeated injection”
NO.
r_-Rhamnose D-i%COSe
1
0.439
2 3 4 5 6
0.462 0.458 0.428 0.452 0.453 0.449 0.012
Mean Standard deviation Standard vaIueb
0.440
L-Arabinose D-XyIose
D-Mannose D-Galacfose D-Gkose
0.444 0.444 0.439
0.367 0.364 0.009
0.422 0.445 0.441 0.439 0.008
0.550 0.546 0.575 0.534 0.564 0.559 0.555 0.013
0.644 0,655 0.660 0.631 0.661 0.668 0.653 0.012
0.563 0.580 0.579 0.555 0.578 0.587 0.574 0.011
0.789 0.760 0.802 0.808 0.807 0.815 0.797 0.018
0.350
0.423
0.546
0.654
0.599
0.800
0.351 0.367 0.377 0.353 0.369
aRatio of the area of 1 pg of sugar to the area of 1 pg of myo-inositol, obtained by repeated injection of l-p1 samples containing all sugars as their alditol acetates (see text). ?he standard value represents the mean of 13 such samples injected at least 5 times each.
D-galactose, D-rnannose, and D-xylose were purchased from Sigma Chemical Company, L-rhamnose from Pfanstiehl Laboratories, and D-glucose from J. T. Baker & Co.; these were found to contain less than 1% of monosaccharide contaminants. myo-Inositol (California Biochemical Corporation) was recrystallized 3 times from ethanol-water and contained no detectable contamination. The detector response was found to be a linear function of the amount of sugar injected over the range of 0.25 to 3 pg. Amounts of sugar above this range (as high as 12 pg) were also accurately determined. Each sample was injected repeatedly into 2 different FM gas chromatographs. The data are presented in Table I as the ratio of the area obtained for 1 ,~g of sugar to that for 1 pg of myo-inositol. The mean values obtained for the various sugars of this sample deviate only slightly from the standard values presented on the bottom line. The major advantages of this procedure over the methods previously reported6’8 are: (1) The trifiuoroacetic acid used in the hydrolysis of polysaccharides is readily removed by evaporation. (2) The entire procedure may be performed in a single test-tube (a second test-tube is required when the hydrolyzed material contains an insoluble residue; this necessitates a single tranfer). (3) Acetylation of the alditols is catalyzed by the sodium acetate present, thus eliminating the need for pyridine. Hence, the acetylation mixture may be injected directly into the gas chromatograph. (4) Acetylation of the alditols is performed in a sealed tube, which eliminates the need for heating under reilux. (5) The column material described gives a better separation of the Carbohyd. Res., 5 (1967) 340-345
345
NOTES
derivatives of the sugars obtained from plant cell-walls than do commercial preparations currently available. ACKNOWLEDGMENTS
This investigation was supborted, in part, by Contract #AT (1 l-1)-1426 with the U. S. Atomic Energy Commission, and by a gram from the Council of Creative Work and Research, University of Colorado. The expert electrical engineering advice and assistance of John Cowan, and the assistance of Thomas M. Jones in preparing this manuscript are gratefully acknowledged. Department of Chemistry,
-
University of Colorado,
DONALD
Boulder, Colorado 80302 &J. S. A.)
f%BERSHEIM
PATRICIA
J. NEVINS D.
ENGLISH
ARTHURKARR REFERENCES
1 J. P. THORNJSERAND D.H.NORTHCOTE, 2 3 4 5 6 7 8 9 10
Biochem. J., 81(1961)455. P. M. RAY, Biochem. J., 89 (1963) 144. G. E. BECKER, P. A. Hur, AND P. ALBERSHEIM,Plant Physiol., 39 (1964) 913. P. M. RAY AND D. B. BAKER, PIanf Physiol., 40 (1965) 353. S. W. GUNNER, J. K. N. JONES, AND M. B. PERRY, Chem. Ind. (London), (1961) 255. J. S. SAWARDEKER,J. H. SLONEKER.AND A. R. JEANES,Anal. Chem., 37 (1965) 1602. E. S&km&I, P. HACLUND, AND J. JANSON, Svensk Papperstidn., 69 (1966) 381. E. P. CROWELL AND B. B. BURNE-I-~,Anal. Chem., 39 (1967) 121. W. T. DIBLE, E. TRUOG, AND K. C. BERGER, Anal. Chem., 26 (1954) 418. D. T. A. LASWORT, Exprl. CeN &es., 33 (1964) 195.
(Received February 6th, 1967; in revised form, July 5th, 1967). Carbohyd. Res..
5 (1967) 340-345
NOTES
for
polysaccharides
the
analysis
of sugars
by gas-liquid
in plant
cell-wall
chromatography
Numerous attempts have been made to determine the sugar composition of plant cell-wall polysaccharides 1--4 by laborious methods which are subject to large experimental errors. Apromising approach to the analysis of monosaccharides involves the conversion of the sugars into their respective alditol acetates, which are then separated by gas-liquidchromatographySy6. This method has been used for analyzing the acid hydrolyzates of polysaccharides 7v8, but, although it has several advantages over earlier methods of polysaccharide analysis, it is unsuitable for analysis of a large number of samples. Modifications that permit rapid and reproducible analysis of the sugar constituents of polysaccharides of plant cells are here described. Trifluoroacetic acid was used for hydrolyzing the polysaccharides, because (a> the yields of most monosaccharides from plant cell-wall preparations are at least equal to those obtained by hydrolysis with mineral acids, and (b) it may be readily removed by evaporation. Fig. 1 shows the yields of sugars obtained from cell walls of pinto-bean hypocotyls on hydrolysis with 2~ trifluoroacetic acid in a sealed tube at 121o for various periods of time. In general, 2~ trifluoroacetic acid (2 ml) was added to the wall material (20 mg); myo-inositol (1 mg) was added as an internal standard, as it is not present in cell walls and has a retention time quite different from those of the various sugars of
8r
TIME
(HOURS)
Fig. 1. Infiuence of the length of hydrolysis on the determination of the carbohydrate component8 of pinto-bean hypocotyl cell-walls. The cell walls were hydrolyzed with 2~ trifluoroacetic acid at 121 o for various periods of time.
Cprbohyd.Res., 5 (1967) 340-345
341
NOTES
the wall. ,4fter hydrolysis for 1 h (optimal for most of the sugars), the soluble portion was evaporated to dryness at 50” under a stream of Altered air. The sugar mixtures obtained from hydrolyzed polysaccharides were reduced to their respective alditols with sodium borohydride (10 mg) in N ammonia (OS ml). The ammonia was added to ensure that those solutions that contained uranic acids and/or traces of trifluoroacetic acid were alkaline during reduction. After 1 h at room temperature (found sufficient for complete reduction of aldoses), the excess of borohydride was decomposed by dropwise addition of glacial acetic acid, un&il effervescence had ceased_ The borate produced on decomposition of the borohydride forms a complex with the alditols, and retards their acetylation. It was found that removal of boric acid improved the acetylation. A mixture of aldoses (rhanmose, xyiose, and glucose) and alditols (arabinitol and mannitol) was subjected to the reduction. To aliquots (1 ml) of this mixture in test tubes, 1 to 8 additions of methanol (1 ml) were made, each followed by evaporation to dryness at 50” under a stream of filtered air. Acetic anhydride (1 ml) was then added and the tubes were sealed and heated for 1 h at 100”. It is evident from Fig. 2 that a stigle addition of methanol had little effect on the degree -8
-7
-6
%
-6
s 9
-4
n b LI
-3
ii! ii? %
t 2
kl_____. , , 0
1
2
3
Additions
4
5
of
methanol
6
1
s
Fig. 2. Influence of the number of additions of methanol on the degree of acetylation. Aliquots (1 ml) were transferred from a mixture (10 ml) containing 1 mg of L-rhamnose, L-arabinitol, r+xylose, D-mannitol, and D-glucose per ml into each of 9 test tubes. These samples were reduced with N ammonia (0.5 ml) containing sodium borohydride (10 mg) and then subjected to various numbers of sequential additions of 1 ml of methanol with evaporation to dryness after each addition. The number of additions of methanol differed from tube to tube. The samples were acetylated, and assayed. as described in the text.
Carbohyd. Rcs., 5 (1967) 340-345
342
NOTES
to which the alditols were acetylated (see below for details on chromatography). However, additional methanol evaporations, up to a total of 4, increased the degree of acetylation. Quantitative analysis of the boric acid remaining after each evaporation’ showed that 5 additions and evaporations of methanol gave optimal results. Acetylation of the alditols was performed in a sealed tube with acetic anhydride (1 ml) heated for 3 h at 121”. The sodium acetate remaining after the removal of boric acid (as its trimethyl ester) served as the basic catalyst for the acetylation reaction. Pyridine, used for this purpose by previous workers, had a deleterious effect on the column material and produced severe tailing of the peaks, especially of the solvent peak. The best g.1.c. separation was obtained with a liquid phase containing 0.2% of poly(ethylene glycol succinate), 0.2% of poly(ethylene glycol adipate) and 0.4% of silicone XFl150 coated on Gas-Chrom P (100-120 mesh) (Applied Science Labs., State College, Pa.). The dried material (ca. 1.1 g) was packed by vibration into a copper tube (4 ft x l/8 in O.D.). A balancing column (5 ft long), containing about 1.4g of the same material, was similarly packed. The columns were conditioned at 180” for 4 h. Separations with this column material were superior to those previously reported 6g8. Fig. 3 illustrates the response of a 5.0-mV recorder to a l-,~l injection of an acetic anhydride solution containing 1 pg of each of the following: the pentaacetates -SO-
-4.0Y >
Rha
-5 z
P
-3.o-
% k z E
-2.o-
,o 8 ii -l.O-
-
0 5 1 TEMPERATURE
(‘C.)
Fig. 3. Mustration of the recorder response for a l-_A injection of acetic anhydride containing 1 pg each of L-rhamnose, D-fucose, L-arabinose, D-xylose, D-mannose, D-galactose, D-glucose, and myo-inositol after reduction and acetylation. The F & M gas chromatograph electrometer \vas set at range 10 and attenuation 1. Full-scale response on the strip-chart recorder WE+5.0 mV. The injcction-port temperature was 210”, and the detector temperature was 250”. The column temperature on injection was 120’; this was maintained for 10 min after injection, and then raised by lo per min. Carbohyd. Res., 5 (1967)
340-345
NOTES
343
of arabinitol, fucitol, rhamnitol, and xylitol, and the hexaacetates of galactitol, glucitol, mannitol, and myo-inositol. The sensitivity achieved in this procedure (see Fig. 3) was obtained by setting the electrometer at range 10 and the attenuation at 1. The area under each peak was measured by electronic- integration. The output of the gas chromatographs was recorded on instrumentation-quality magnetic tape (Scotch Brand X 860, Minnesota Mining and Manufacturing Co.) with an Infotronics Model CRS-43RlD magnetic tape recorder, and was monitored by means of an Infotronics Model CRS-40TS playback system at 16 times the recording speed (7.5 in/set). Integration of the response peaks was achieved with an Infotronics digital readout system Model CRS-1 lHS/42. Results obtained in the analysis of cell-wall polysaccharides are given in Fig. 4. The cell walls analyzed in this experiment were obtained from sycamore (Acer pseudoplatanus) cells grown in suspension culture’o on a defined (MID) medium*. A sample of cell wall (16 mg) was hydrolyzed, and reduced, and then acetylated with acetic anhydride (1 ml). One 1.11was injected into the gas chromatograph to give the recorder responses shown in Fig. 4. The accuracy and precision of this method of analysis were tested as follows (Table I): samples, each containing individually weighed amounts of the 7 sugars -5.0
-
-4.o7 > .5 :
--3.o-
% % 2
Rha -*o-
5 2 8 z
- 1.0-
-o1 TEMPERATURE
(‘C.)
Fig- 4. Gas-chromatographic analysis ofa I+1 injection containing the material from sycamore (Acer pse&q&tanus) cell-walls. The cell walls were hydrolyzed, and the products reduced, acetylated, and analyzed as described in the text. *Personal communication from Philip Filner, Michigan State University. Carbohyd. Res., 5 (1967) 340-345
344
NOTES
found in plant cell-walls and myo-inositol (1 mg/ml), were subjected to acid hydrolysis, reduction, acetylation, and assay by gas chromatography. L-Arabinose, D-fucose, TABLE PRECISION
Injecrion
I AND
ACCURACY
OF THE
METHOD
Proportion of sugar components after repeated injection”
NO.
r_-Rhamnose D-i%COSe
1
0.439
2 3 4 5 6
0.462 0.458 0.428 0.452 0.453 0.449 0.012
Mean Standard deviation Standard vaIueb
0.440
L-Arabinose D-XyIose
D-Mannose D-Galacfose D-Gkose
0.444 0.444 0.439
0.367 0.364 0.009
0.422 0.445 0.441 0.439 0.008
0.550 0.546 0.575 0.534 0.564 0.559 0.555 0.013
0.644 0,655 0.660 0.631 0.661 0.668 0.653 0.012
0.563 0.580 0.579 0.555 0.578 0.587 0.574 0.011
0.789 0.760 0.802 0.808 0.807 0.815 0.797 0.018
0.350
0.423
0.546
0.654
0.599
0.800
0.351 0.367 0.377 0.353 0.369
aRatio of the area of 1 pg of sugar to the area of 1 pg of myo-inositol, obtained by repeated injection of l-p1 samples containing all sugars as their alditol acetates (see text). ?he standard value represents the mean of 13 such samples injected at least 5 times each.
D-galactose, D-rnannose, and D-xylose were purchased from Sigma Chemical Company, L-rhamnose from Pfanstiehl Laboratories, and D-glucose from J. T. Baker & Co.; these were found to contain less than 1% of monosaccharide contaminants. myo-Inositol (California Biochemical Corporation) was recrystallized 3 times from ethanol-water and contained no detectable contamination. The detector response was found to be a linear function of the amount of sugar injected over the range of 0.25 to 3 pg. Amounts of sugar above this range (as high as 12 pg) were also accurately determined. Each sample was injected repeatedly into 2 different FM gas chromatographs. The data are presented in Table I as the ratio of the area obtained for 1 ,~g of sugar to that for 1 pg of myo-inositol. The mean values obtained for the various sugars of this sample deviate only slightly from the standard values presented on the bottom line. The major advantages of this procedure over the methods previously reported6’8 are: (1) The trifiuoroacetic acid used in the hydrolysis of polysaccharides is readily removed by evaporation. (2) The entire procedure may be performed in a single test-tube (a second test-tube is required when the hydrolyzed material contains an insoluble residue; this necessitates a single tranfer). (3) Acetylation of the alditols is catalyzed by the sodium acetate present, thus eliminating the need for pyridine. Hence, the acetylation mixture may be injected directly into the gas chromatograph. (4) Acetylation of the alditols is performed in a sealed tube, which eliminates the need for heating under reilux. (5) The column material described gives a better separation of the Carbohyd. Res., 5 (1967) 340-345
345
NOTES
derivatives of the sugars obtained from plant cell-walls than do commercial preparations currently available. ACKNOWLEDGMENTS
This investigation was supborted, in part, by Contract #AT (1 l-1)-1426 with the U. S. Atomic Energy Commission, and by a gram from the Council of Creative Work and Research, University of Colorado. The expert electrical engineering advice and assistance of John Cowan, and the assistance of Thomas M. Jones in preparing this manuscript are gratefully acknowledged. Department of Chemistry,
-
University of Colorado,
DONALD
Boulder, Colorado 80302 &J. S. A.)
f%BERSHEIM
PATRICIA
J. NEVINS D.
ENGLISH
ARTHURKARR REFERENCES
1 J. P. THORNJSERAND D.H.NORTHCOTE, 2 3 4 5 6 7 8 9 10
Biochem. J., 81(1961)455. P. M. RAY, Biochem. J., 89 (1963) 144. G. E. BECKER, P. A. Hur, AND P. ALBERSHEIM,Plant Physiol., 39 (1964) 913. P. M. RAY AND D. B. BAKER, PIanf Physiol., 40 (1965) 353. S. W. GUNNER, J. K. N. JONES, AND M. B. PERRY, Chem. Ind. (London), (1961) 255. J. S. SAWARDEKER,J. H. SLONEKER.AND A. R. JEANES,Anal. Chem., 37 (1965) 1602. E. S&km&I, P. HACLUND, AND J. JANSON, Svensk Papperstidn., 69 (1966) 381. E. P. CROWELL AND B. B. BURNE-I-~,Anal. Chem., 39 (1967) 121. W. T. DIBLE, E. TRUOG, AND K. C. BERGER, Anal. Chem., 26 (1954) 418. D. T. A. LASWORT, Exprl. CeN &es., 33 (1964) 195.
(Received February 6th, 1967; in revised form, July 5th, 1967). Carbohyd. Res..
5 (1967) 340-345