- Email: [email protected]
Hemicellulose fractions of some hays and straws
Hemicellulose
Fractions
of Some Hays and Straws1
C. A. Flanders’ b’~om
the Department
of Agriculturdl Morgantown, Received
Biochemistry,
Shyest l’irginia
~~‘niversil~~,
West Virginia September
17, 195 I
Most of the workers attempting to fractionate the hemicelluloses have followed the example of O’Dwyer (5) and ext.racted the hemicelluloses as a whole and then precipitated the first fraction with acid (A fraction) and subsequent fractions by the addition of increasing amounts of alcohol or acetone. A few have subdivided these fractions. Bennett (1) applied solvents of different alkaline strengths to cornstalks and obtained hemicellulose fractions which were significantly different. This investigation deals with the successive extraction of hemicellulose fractions by means of solvents of increasing alkalinity from the chlorite holocelluloses of alfalfa and red clover hays and of barley, buckwheat, oat, and wheat straws. While the hemicelluloses of some of these plant materials have been isolated, a study of these hemicellulose fractions has not been previously reported. PROCEDURE The red clover and alfalfa hays were cut in the early bloom stage. The straws, essentially free of chaff, were secured after threshing from grains cut when the grain was ripe. All the samples were ground in a Wiley mill until they would pass through a lo-mesh sieve. Starting with the ground plant material the procedure for the isolation of the hemicellulose fractions is that described by the author (3) in a previous publication. Pentoses were determined by the method of McRary and Slattery (4) and corrected for the presence of uranic acid. The uranic acid anhydride was determined 1 Based upon a dissertation submitted in partial fulfillment of the requirements of West Virginia University for the degree of Doctor of Philosophy. Published with the approval of the Director of the West Virginia Agricultural Experiment Station as Scientific Paper No. 447. 2 Present address: Fisher Scientific Company, 685 Grrenwich Street, Kcw York 14, N. Y.
425
426
C.
by the method of Browning weighing the residue.
A.
FLANDERS
(2). Ash was determined
by ignition
of a sample and
RESULTS
Table I shows the yield of holocellulose obtained from the extracted plant material. The extracted plant material and the holocelluloses TABLE Yields
of
I
Holocellulose on Basis of h’xtracted Plant
Plant
Material
HOlOCEllulOS~ yield 0
material
Alfalfa hay Red clover hay Barley straw Buckwheat straw Oat straw Wheat straw
Ezo 77.5 78.7 69.7 69.6 79.4
QCorrected for ash.
corrected for ash by ignition, but no attempt was made to correct the holocellulose for residual lignin. Table II shows the hemicellulose yield for each fraction obtained from each of the holocelluloses.
were
TABLE Per Cent
Yield
of Hemicellulose
II Fractions
from
Hobcellulose
All values corrected for ash Solvent
2.5% K&OS 5% KOH 10% KOH 200/, KOH
Alfalfa
hay
10.9 a.7 9.3 5.2
Redh:?
14.5 7.0 12.4 8.6
Barley straw
16.8 14.8 14.5 4.6
Buckwheat straw
8.3 11.7 18.8 8.8
oat straw
9.5 6.8 9.7 4.5
Wheat straw
11.4 13.9 16.6 5.1
Before one makes comparisons between these plant sources, it should be noted that there are three plant families represented. Alfalfa and red clover are legumes; barley, oats, and wheat are grasses; while buckwheat is a member of the buckwheat family. From the data in Table III it will be seen that in all cases the per cent of uranic acid anhydride decreased as the alkalinity of the solvent was increased. The legumes showed the highest uranic acid content in all their fractions. Buckwheat hemicelluloses showed a uranic acid anhydride content intermediate between that of the legumes and the grasses.
HEMICELLULOSE
TABLE Per Cent Solvent
Uranic
Acid
Anhydride
Redhz=
28 17.8 13.3 10.4
III of Hemicellulose Barley straw
Alfalfa
2.5% K&03 5% KOH 10% KOH 2091, KOH
127
FRACTIONS
30.4 26.5 14.6 13.0
Fractions
Buckwheat straw
13.2 8.4 6.5 5.6
oat straw
22.1 13.1 10.8 8.7
13.0 8.7 5.5 .5. I
Wheat straw
9.7 7.0 5.4 5.0
The pentose content of the hemicellulose fractions (Table IV> did not show the regularity of change from fraction to fraction shown by the uranic acid content. All of the grasses showed a regular increase in pent,ose content in the first three fractions, and all of the grasses TABLE Per Cent Pentose Solvent
2.5% KzCOs 5yo KOH 10% KOH 20% KOH
Alfalfa hay
Red
34.1 64.3 60.8 68.9
IV
of the Hemicelldose clover
Fractions
bY
Barley straw
Buckwheat straw
Oat straw
Wheat straw
22.5 51.9 60.9 61.4
66.8 71.0 78.0 69.2
64.1 62.1 75.!) 6X.7
74.7 76.1 76.2 73.8
71.1 76.8 75.5 72.9
showed a slight drop in pentose content from the third to the last fraction. Buckwheat also showed this decrease in pentose content for the last fraction. The two legumes were not consistent. The pentose content of the red clover increased regularly, while in the case of the alfalfa the pentose content of the second fraction was higher than that of the third fraction. Neither legume showed t,he decrease in pentose content from the third to the fourth fraction t,hat was found in the grasses. The pentose/uronic acid anhydride ratios increased from t,he 5yc K&O3 fraction through the 20y0 KOH fraction. However, as can be seen in Table V, these differences in several cases are very small, Also, TABLE Pentose/Uronic so1\wlt
5% K&O3 2.5% KOH 10% KOH 2070 KOH
Acid
Anhyriok
Alfalfa
Molecular Red
olover
hay
&Y
1.1 4.2 5.4 7.i
1.2 2.3 4.8 5.0
V Ratios Barley straw
6.0 9.9 14.0 14.5
in the Hemicellulose Buckwheat straw
3.4 5.5 8.2 8.7
Fractionn
oat straw
Wheat straw
6.i 10.2 16.1 17.0
8.5 12.9 16.4 18.1
228
C.
A.
FLANDERG
there appears to be no relation between the ratio in a fraction from one source and the ratio found in the hemicellulose of the same fraction from another source. From this it must be concluded that the fractions are mixtures of molecules of varying pentose/uronic acid anhydride content with a varying degree of ease of separation from the holocellulose. These ratios are really weighted averages of the ratios in the hemicelluloses of the mixtures, the averages being determined by the proportion of high or low ratios in the range covered. In several cases the preponderance of either high ratios in the 10% KOH fraction range or more probably the accumulation of lower ratios in the 20% KOH fraction, causes these last two fractions to have pentose/uronic acid anhydride ratios which are not very far apart. The reasons for attributing this effect to the 20% KOH are twofold. First, the effect of increasing alkalinity on solubility has almost reached its peak at the 10% level. Second, the difference between the alkalinity of the 2.50/c and the 10% KOH is 0.3 pH units greater than between the 10 and 20% KOH. The primary reason for using the 20% KOH is to dissolve the last of the hemicellulose removable by an alkaline solvent. Another factor affecting these ratios lies in the probable presence of some nonuronide hemicellulose molecules mixed with polyuronide hemicellulose molecules in the same fraction, as the result of having long-chain molecules containing uranic acid with the same solubility as short-chain molecules containing no uranic acid. Now, if one examines Table V for the ratios of molecules of pentose per molecule of uranic acid anhydride as they vary from plant to plant, it will be seen that the legumes showed the smallest molecular ratios, while buckwheat was intermediate between the legumes and the grasses. The ratio for the 20% KOH fraction of the legumes was about the starting point for the 5$‘/& K&OS fraction of the grasses. Apparently the primary effect of this fractionation by differential solvents was to set up arbitrary fractions. More fractions can be obtained by varying the degree of alkalinity, the only difficulty being that from a practical standpoint the amount of precipitate is apt to be too small to handle properly. The contribution each hemicellulose fraction makes to the total yield of hemicelluloses varies considerably from the holocellulose of one plant material. to another plant holocellulose. This can be seen in Table VI which gives the per cent of the total hemicelluloses found in each fraction from different plants. From this table it can be seen that
HEMICELLULOSE
TnBLE I'er Solvent
5%&C03 2.5% KOH 10% KOH 20%, KOH
Cent of Total
429
FRACTIONS
VI
Hemicellulose
I”oand
Alfalfa
Red clover
Iladey
hay
hay
straw
32.0 25.5 27.3 15.2
34.0 16.5 29.2 20.2
33.0 2Q.2 28.6 !I. 1
in Bach Praclion Buckwheat straw
17.4 24.6 39.5 18.5
oat straw
31.0 22.3 :!l1.t3 14.8
Wheat straw
24.3 27.4 35.3 lo.!)
with few exceptions the 5y0 KzC03 solution yielded the most hemicelluloses, the 10% KOH is next, the 2.5$$ KOH third, and the 20yc, KOH solution yielded least hemicelluloses. SUMMARY
Chlorite holocelluloses were prepared from two hays (alfalfa and red clover) and four straws (barley, buckwheat, oat, and wheat). By means of alkaline solvents of increasing alkalinity, four hemicellulose fractions were extracted from each of the six holocelluloses. The hemicellulose fractions were analyzed and the ratios of molecules of pentose units per molecule of uranic acid anhydride were determined. The ratio of pentose to uranic acid anhydride molecules increased as the alkalinity of the solvent increased. The fractions secured were mixtures and their over-all composition varied from one plant source to another. The uranic acid anhydride and pentose content of the hemicellulose fractions showed comparative variations within plant families but not between plants of different families. Buckwheat hemicelluloses fill an intermediate position between those of the legumes and the grasses in uranic acid anhydride content, pentose content, pentose/uronic acid anhydride ratios, and changes in these values from one hemicellulose fraction to another. REFERENCES
BENNETT, E., Arch. Biochem. 27, 99 (1950). BROWNING, B. L., TAPPI 32, 119 (1949). 3. FLANDERS, C. A., Arch. Biochem. Biophys. 36, 421 (1952). 4. MCRARY, N7. L., AND SLATTERY, M. C., Arch. Biochem. 6, 151 (1945). 1.
2.
5. O’DWYER,
M.
H., Biochem.
J. 7, 501 (1923).
Fractions
of Some Hays and Straws1
C. A. Flanders’ b’~om
the Department
of Agriculturdl Morgantown, Received
Biochemistry,
Shyest l’irginia
~~‘niversil~~,
West Virginia September
17, 195 I
Most of the workers attempting to fractionate the hemicelluloses have followed the example of O’Dwyer (5) and ext.racted the hemicelluloses as a whole and then precipitated the first fraction with acid (A fraction) and subsequent fractions by the addition of increasing amounts of alcohol or acetone. A few have subdivided these fractions. Bennett (1) applied solvents of different alkaline strengths to cornstalks and obtained hemicellulose fractions which were significantly different. This investigation deals with the successive extraction of hemicellulose fractions by means of solvents of increasing alkalinity from the chlorite holocelluloses of alfalfa and red clover hays and of barley, buckwheat, oat, and wheat straws. While the hemicelluloses of some of these plant materials have been isolated, a study of these hemicellulose fractions has not been previously reported. PROCEDURE The red clover and alfalfa hays were cut in the early bloom stage. The straws, essentially free of chaff, were secured after threshing from grains cut when the grain was ripe. All the samples were ground in a Wiley mill until they would pass through a lo-mesh sieve. Starting with the ground plant material the procedure for the isolation of the hemicellulose fractions is that described by the author (3) in a previous publication. Pentoses were determined by the method of McRary and Slattery (4) and corrected for the presence of uranic acid. The uranic acid anhydride was determined 1 Based upon a dissertation submitted in partial fulfillment of the requirements of West Virginia University for the degree of Doctor of Philosophy. Published with the approval of the Director of the West Virginia Agricultural Experiment Station as Scientific Paper No. 447. 2 Present address: Fisher Scientific Company, 685 Grrenwich Street, Kcw York 14, N. Y.
425
426
C.
by the method of Browning weighing the residue.
A.
FLANDERS
(2). Ash was determined
by ignition
of a sample and
RESULTS
Table I shows the yield of holocellulose obtained from the extracted plant material. The extracted plant material and the holocelluloses TABLE Yields
of
I
Holocellulose on Basis of h’xtracted Plant
Plant
Material
HOlOCEllulOS~ yield 0
material
Alfalfa hay Red clover hay Barley straw Buckwheat straw Oat straw Wheat straw
Ezo 77.5 78.7 69.7 69.6 79.4
QCorrected for ash.
corrected for ash by ignition, but no attempt was made to correct the holocellulose for residual lignin. Table II shows the hemicellulose yield for each fraction obtained from each of the holocelluloses.
were
TABLE Per Cent
Yield
of Hemicellulose
II Fractions
from
Hobcellulose
All values corrected for ash Solvent
2.5% K&OS 5% KOH 10% KOH 200/, KOH
Alfalfa
hay
10.9 a.7 9.3 5.2
Redh:?
14.5 7.0 12.4 8.6
Barley straw
16.8 14.8 14.5 4.6
Buckwheat straw
8.3 11.7 18.8 8.8
oat straw
9.5 6.8 9.7 4.5
Wheat straw
11.4 13.9 16.6 5.1
Before one makes comparisons between these plant sources, it should be noted that there are three plant families represented. Alfalfa and red clover are legumes; barley, oats, and wheat are grasses; while buckwheat is a member of the buckwheat family. From the data in Table III it will be seen that in all cases the per cent of uranic acid anhydride decreased as the alkalinity of the solvent was increased. The legumes showed the highest uranic acid content in all their fractions. Buckwheat hemicelluloses showed a uranic acid anhydride content intermediate between that of the legumes and the grasses.
HEMICELLULOSE
TABLE Per Cent Solvent
Uranic
Acid
Anhydride
Redhz=
28 17.8 13.3 10.4
III of Hemicellulose Barley straw
Alfalfa
2.5% K&03 5% KOH 10% KOH 2091, KOH
127
FRACTIONS
30.4 26.5 14.6 13.0
Fractions
Buckwheat straw
13.2 8.4 6.5 5.6
oat straw
22.1 13.1 10.8 8.7
13.0 8.7 5.5 .5. I
Wheat straw
9.7 7.0 5.4 5.0
The pentose content of the hemicellulose fractions (Table IV> did not show the regularity of change from fraction to fraction shown by the uranic acid content. All of the grasses showed a regular increase in pent,ose content in the first three fractions, and all of the grasses TABLE Per Cent Pentose Solvent
2.5% KzCOs 5yo KOH 10% KOH 20% KOH
Alfalfa hay
Red
34.1 64.3 60.8 68.9
IV
of the Hemicelldose clover
Fractions
bY
Barley straw
Buckwheat straw
Oat straw
Wheat straw
22.5 51.9 60.9 61.4
66.8 71.0 78.0 69.2
64.1 62.1 75.!) 6X.7
74.7 76.1 76.2 73.8
71.1 76.8 75.5 72.9
showed a slight drop in pentose content from the third to the last fraction. Buckwheat also showed this decrease in pentose content for the last fraction. The two legumes were not consistent. The pentose content of the red clover increased regularly, while in the case of the alfalfa the pentose content of the second fraction was higher than that of the third fraction. Neither legume showed t,he decrease in pentose content from the third to the fourth fraction t,hat was found in the grasses. The pentose/uronic acid anhydride ratios increased from t,he 5yc K&O3 fraction through the 20y0 KOH fraction. However, as can be seen in Table V, these differences in several cases are very small, Also, TABLE Pentose/Uronic so1\wlt
5% K&O3 2.5% KOH 10% KOH 2070 KOH
Acid
Anhyriok
Alfalfa
Molecular Red
olover
hay
&Y
1.1 4.2 5.4 7.i
1.2 2.3 4.8 5.0
V Ratios Barley straw
6.0 9.9 14.0 14.5
in the Hemicellulose Buckwheat straw
3.4 5.5 8.2 8.7
Fractionn
oat straw
Wheat straw
6.i 10.2 16.1 17.0
8.5 12.9 16.4 18.1
228
C.
A.
FLANDERG
there appears to be no relation between the ratio in a fraction from one source and the ratio found in the hemicellulose of the same fraction from another source. From this it must be concluded that the fractions are mixtures of molecules of varying pentose/uronic acid anhydride content with a varying degree of ease of separation from the holocellulose. These ratios are really weighted averages of the ratios in the hemicelluloses of the mixtures, the averages being determined by the proportion of high or low ratios in the range covered. In several cases the preponderance of either high ratios in the 10% KOH fraction range or more probably the accumulation of lower ratios in the 20% KOH fraction, causes these last two fractions to have pentose/uronic acid anhydride ratios which are not very far apart. The reasons for attributing this effect to the 20% KOH are twofold. First, the effect of increasing alkalinity on solubility has almost reached its peak at the 10% level. Second, the difference between the alkalinity of the 2.50/c and the 10% KOH is 0.3 pH units greater than between the 10 and 20% KOH. The primary reason for using the 20% KOH is to dissolve the last of the hemicellulose removable by an alkaline solvent. Another factor affecting these ratios lies in the probable presence of some nonuronide hemicellulose molecules mixed with polyuronide hemicellulose molecules in the same fraction, as the result of having long-chain molecules containing uranic acid with the same solubility as short-chain molecules containing no uranic acid. Now, if one examines Table V for the ratios of molecules of pentose per molecule of uranic acid anhydride as they vary from plant to plant, it will be seen that the legumes showed the smallest molecular ratios, while buckwheat was intermediate between the legumes and the grasses. The ratio for the 20% KOH fraction of the legumes was about the starting point for the 5$‘/& K&OS fraction of the grasses. Apparently the primary effect of this fractionation by differential solvents was to set up arbitrary fractions. More fractions can be obtained by varying the degree of alkalinity, the only difficulty being that from a practical standpoint the amount of precipitate is apt to be too small to handle properly. The contribution each hemicellulose fraction makes to the total yield of hemicelluloses varies considerably from the holocellulose of one plant material. to another plant holocellulose. This can be seen in Table VI which gives the per cent of the total hemicelluloses found in each fraction from different plants. From this table it can be seen that
HEMICELLULOSE
TnBLE I'er Solvent
5%&C03 2.5% KOH 10% KOH 20%, KOH
Cent of Total
429
FRACTIONS
VI
Hemicellulose
I”oand
Alfalfa
Red clover
Iladey
hay
hay
straw
32.0 25.5 27.3 15.2
34.0 16.5 29.2 20.2
33.0 2Q.2 28.6 !I. 1
in Bach Praclion Buckwheat straw
17.4 24.6 39.5 18.5
oat straw
31.0 22.3 :!l1.t3 14.8
Wheat straw
24.3 27.4 35.3 lo.!)
with few exceptions the 5y0 KzC03 solution yielded the most hemicelluloses, the 10% KOH is next, the 2.5$$ KOH third, and the 20yc, KOH solution yielded least hemicelluloses. SUMMARY
Chlorite holocelluloses were prepared from two hays (alfalfa and red clover) and four straws (barley, buckwheat, oat, and wheat). By means of alkaline solvents of increasing alkalinity, four hemicellulose fractions were extracted from each of the six holocelluloses. The hemicellulose fractions were analyzed and the ratios of molecules of pentose units per molecule of uranic acid anhydride were determined. The ratio of pentose to uranic acid anhydride molecules increased as the alkalinity of the solvent increased. The fractions secured were mixtures and their over-all composition varied from one plant source to another. The uranic acid anhydride and pentose content of the hemicellulose fractions showed comparative variations within plant families but not between plants of different families. Buckwheat hemicelluloses fill an intermediate position between those of the legumes and the grasses in uranic acid anhydride content, pentose content, pentose/uronic acid anhydride ratios, and changes in these values from one hemicellulose fraction to another. REFERENCES
BENNETT, E., Arch. Biochem. 27, 99 (1950). BROWNING, B. L., TAPPI 32, 119 (1949). 3. FLANDERS, C. A., Arch. Biochem. Biophys. 36, 421 (1952). 4. MCRARY, N7. L., AND SLATTERY, M. C., Arch. Biochem. 6, 151 (1945). 1.
2.
5. O’DWYER,
M.
H., Biochem.
J. 7, 501 (1923).