1 38 Tech Bull Fac Agr Kagawa Univ
ISOLATION AND DETERMINATION OF SUGARS FROM THE COTYLEDON,
HULL, AND HYPOCOTYL OF SOYBEANS BY CARBON
COLUMN CHROMATOGRAPHY
Sin'itirG KAWAMURA
and
Minoru TADA*
Introduction
The data on sugar determinations in the preceding papers(' 2, were obtained almost exclu- sively by quantitative paper chromatography. Thus i t is desired to make some experiments on the isolation and identification of such sugars, though i t is now established t h a t the oligosaccha~ides of soybeans are mainly sucrose, raffinose, and stachyose
.
The separation and identification of sugars with carbon column chromatography were carried on the three parts of soybeans, i . e. cotyledon, hull, and hypocotyl.Materials and Methods
As the sample the cotyiedon, hull, and hypocotyl were used which were separated from the Harosoy soybeans .('I
In principle the method of WHISTLER and
I ) u ~ s o ( ~ )
was followed. The adsorbent was a mixture of 9 g Darco G 60 and 9 p: Celite 535, kneaded with 50% ethanol. I t was put in a column of 350 mm long and 18 mm diameter and was washed with 50% ethanol and then with water.Results and Discussion
Preliminary experiments showed that water eluted glucose, 5
%
ethanol eluted sucrose, and 10 % ethanol eluted raffinose, the recoveries being 92.1, 99.3, and 84.6 %, respec- tively. The higher the sugar i s , the longer is the time needed t o collect the fractions. Determination of sugar was easily made by the phenol-sulfuric acid method.Here a n example of quantitative determination of separate sugars is reported on the hull and the defatted cotyledon and hypocotyl from Harosoy soybeans. The result i s shown in Tables 1 .- 3 and Figs. 1 - 3 .
Table 1 Column chromatography of sugars from the defatted cotyledon of Harosoy soybeans Solvent Vol of eluate collected Sugar eluted Amt of sugar
Water I00 ml Arabinose 0 073 mg
Water 400 Glucose 5 69
55% EtOH 980 Sucrose 23 12
10% EtOH 200 Raf f inose 1 61
10-1 5% EtOH 860 Stachyose 19 15
20% EtOH 80 Verbascose 0 084
% on dry Result of paper basis chromatography(') 0 019 .-
I
.59 - 6.47 6.2 0.45 0 8 5.36 4.0 0.023 -*
PI esent addr ess : Kagawa-ken Meizen .Junior College, TakamatsuOLIVE 香川大学学術情報リポジトリ
Vol 18, No 2 (1967)
Cotyledon
Fig. 1 Dete~mination of sugars by column chromatog~aphy (1) Table 2 Column chromatography of sugars from the hull of Harosoy soybeans
Solvent Vol of eluate collected Sugar eluted Amt of sugar % on dry Result of paper basis chromatography(l)
Water I00 ml Ar abinose 0 120 mg 0 069 0 01
Water 100 Gulcose 0 118 0 068 0 05
5 % EtOH 320 Sucrose 5 096 0 29 0 55
10% EtOH 240 Raf f inose 0 762 0 044 0 13
15% EtOH 590 Stachyose 6 42 0 37 0 40
20% EtOH 40 Ver bascose 0 036 0 002 -
I
;1
1.91 Hull mg 1. 2 1 0 - 7 0 8- 0. 6- 0 4- 0 2-1
'
oh
- F r a c . N o . 0 10 20 30 40 50 60 70 80 E t O H Concn I+-Hz0-+rt 5 +I+ 10 +I+ 15 -lc20+5 % -
I I 'I' 4 1-~ r a < -~ l c " ' " SUC. " Raf "' S t a Ver Fig 2 Determination of sugars by column chromatography (21
140 Tech. Bull. Fac
.
Agr.
Kagawa Univ. Table 3 Column chromatography of sugars from the defatted hypocotyl of Harosoy soybeans Solvent Vol of eluate collected Sugar eluted Amt of sugar % on dry Result of paper basis chromatography(1)Water 80 ml Arabinose 0 141 mg 0 048 -
Water 380 Glucose 6 59 0 22 -
5% EtOH 520 Sucrose 30 4 10 3 6 1
10% EtOH 780 Raf finose 37 8 12 8 ? 0 9
12-1 5% EtOH 1000 Stach) ose 4 71 1 6 ? 8 6
20% EtOH 140 Verbascose 0 322 0 11
[HI )It---)l
A r a ( G I c "' S u c "' Raf S t a IF--+ Ver
Fig 3 Determination of sugars by column chromatography (3)
When the results of column chromatography are compared with those of paper chromatog- raphy described in the preceding paper'') the numerical data do not coincide. I n general the column chromatography is taken to be more accurate, since column chromatography gives the result of actual separate colorimetry, while paper chromatography gives the result of approximate colorimetry of spots. However, these determinations were not repeated and the data are tentative.
T h e remarkable discrepancy in case of raffinose and stachyose of hypocotyl (Table 3) may be attributed to erroneous interpretation.. Fig. 3 shows a gap a t fraction number 60 and stachyose might have begun to eluate from this point, though rechromatography was not attempted.
I t should be noted that trace amounts of verbascose were found in all the three parts of Harosoy soybeans. The pentose was tentaively expressed a s arabinose
.
Vol 18, No 2 (1967) 141 Refined sugar solution from 3 0 g hulls was concentrated to 28 ml, 2 m l out of which was put on a column, and 50 fractions per 10 ml were collected. The hull contained arabinose and glucose a s monosaccharides a s revealed by paper chromatography of the concentrated fractions. The arabinose may have been produced by hydrolysis of water-soluble poly- saccharide in t h e hull. I n this connection i t should be noted that the two other parts, cotyledon and hypocotyl also gave the evidence of containing minute amounts of a pentose as revealed by column chromatography.
Summary
A pentose, glucose, sucrose, raffinose, stachyose, and verbascose in the cotyledon, hull, and hypocotyl were separated distinctly by carbon column chromatography. However, the numerical data of some sugars did not coincide with those obtained from quantitative paper chromatogrphy
.
Acknowledgments
This paper corresponds to Section 1 of Chapter 3 (Isolation and identification of the sugars from soybeans) of the final report of research conducted under grants (grant number FG-Ja-105) authorized by Public Law
480
for the Unitzd States Department of Agriculture.References
(1) KAWAMURA, S : Thzs Bulletzn, 18,117 (1967) (3) WHISTLER, R L, DURSO, D F : J Am Chem
(2) KAWAMURA, S : Ibzd
,
132 SOC,
72, 677 (1950) ; 74, 51 40 (1 952)Z
P : % a l p % ~ . f e & & k a a a a B / \ ~ Y 4&3#,
BE,
E%h0
3 K % ' K ~ K & K % ' D & & % ~ ~ ~ ~ ~ ~ ~ A K @4 % 3 @ k k C S , 4~ 1- - 2 , ~ / v T - X , 9 ~ & u - z , 5 7 4 1-2, x Y * ~ - x , ~ 1 ~ / q ~ ~ - ; t f i i 7 k f i a r j ;PIVT-/t/82E%5, 10, 20%kI@&LG;F&K89 T K 7 3 3 k % # L < @ & L < % k L ~ ~ ~ L ? O ~ ; ~ K J B Y % M Z B F J X - ~ T - ~ U T
k
7 9 7 4 - f i ~ b $ ? & k & ~ k & \ b F L % - 5 3 L k f i a 7 kZ D n % M 7 z ! J h E % ~ s % ~ ~ 1 &
(Received Oct 31, 1966)
