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Separation of sugars from soybeans by centrifugal chromatography-香川大学学術情報リポジトリ

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(1)

Tech Bull Fac Agr. Kagawa Univ

SEPARATION OF SUGARS FROM SOYBEANS BY CENTRIFUGAL

CHROMATOGRAPHY

Sin'itirB KAWAMURA,

Hiroshi SUZUKI, and Masakazu IMAYOSI*

Introduction

Quantitative paper chromatography was widely applied in the preceding and carbon column chromatography was also utilized for the isolation of sugars.c3) T h e use of centrifugal chromatography as a rapid method for the separation of sugars is reported i n this paper.

As DEYL et al. in a recent review(4) states, "centrifugal chromatography is i n principle classical chromatography with accelerated flow of the mobile phase due to the action of centrifugl force. Every apparatus for centrifugal chromatography consists of the chamber, an appliance for holding the chromatogaphic paper fixed during rotational movement, and an arrangement for delivering the mobile phase on to t h e paper (distributor of the mobile phase)

."

Apparatus and Method

We used an apparatus of TByB Kagaku Sangy6 K. K . (T6yB Scientific Industries, Ltd.), Tokyo, which is of a n improved McDonald type. The apparatus is depicted in Figure 1. C o n t ~ o l o,f' the influ.x o,f' mobzle phase In the operation of this apparatus, the control of influx of the mobile phase was the most important. The capillary tube of the distributor (micropipette or jet) originally attached to the apparatus a s purchased was not applicable because of a too large amount of influx. At first, thick-wall capillary tube (1 mm diame- ter) of about 1 m long was set in between the micropipette and reservoir of mobile phase so as to decrease the amount of influx based upon the HAGEN-POISEUILLE'S law. The mobile phase flowed i n on filter paper drop by drop in this case and mobile phase developed on the paper only in one direction from the site applied origially to periphery, maybe because of the unevenness of paper. High pressure of about

400

mm Hg or more by compressor was necessary to make the influx a continuous fine stream. This pressure was too high for this apparatus and sometimes rubber stopper on the reservoir of mobile phase slipped out. In order to make a continuous fine stream of a small amount of mobile phase under a relatively low pressure, usual glass tube of

4

mm diameter was used instead of thick- wall capillar y tube and micropipette was made from thick- wall capillary tube by extending so a s to make the diameter about 0.1 mm. Higher pressure was necessary to make the fine stream when the extended capillary micropipette became longer. The smallest speed of influx with fine stream by one of the capillary micropipettes thus made was 0.68 ml/min under a pr essure of 180 mm Hg

.

(2)

Vol 18, No 2 (1967)

Condztzons for operatzon Optimal conditions for separation of each oligosaccha~ide by centr ifugal chromatography we1 e searched concerning the 2 kinds of filter paper, the speed of rotation from 500 to 1700 rpm, the selection of mobile phases, the amount of influx of mobile phases from 0.68 to 1.20ml/ min by increasing the pressure, and the distance of the site of spots applied originally from the center of rotation from 2.5 to 6.0 cm.

TByB No. 514 round filter paper (diameter 32 cm, thickness 0.4 mm) was better than

T6y6 No. 51 UH (thickness 0.2mm), because

k

A

the latter was apt to break when rotated at A. Compressor, rubber with constant pressure

1.500 rpm. For the separation of each spot of r eser voir

oligosaccharide, the rotation speed of more B for pressure chambel C U-Shaped open tube manometer than 1200 rprn was necessary. Each spot was D . Three way stop-cock

apt to tail when butanol-pyridine-water system E. Reservoir of mobile phases was used. The spots of oligosaccharides were ' "Jet of phases

G . Chromatographic chamber separated at the influx of up to 0.78 ml/min

stand

in case of Toy6 No. 514, and up to 0.72 ml/ 1 , Paper support

min in case of Toy6 No. 51 UH. As the Chromatographic paper K Fastening ring micropipette was located at 2.4 cm from the L . " " G ~ ~ ~ ~ lid of center of rotation, separation of each sugar was M

.

Motor

N Revolution - indicator unsuccessful when the original spots were

0 Slidac

applied at more than 5 cm apart from the Fig 1 Apparatus for centrifugal center. The time needed for one development chromatography

was 7-14 min in case of T6y6 No. 514 and

5-9 min in case of T6y6 No. 51 UH, as compared to about 15 hours by ascending chro- matography.

I t was found that the optimal conditions at about 10" (room temperature in winter) were as follows : paper-T6y6 No. 514; rotation--1500 rpm with butanol -acetic acid-water or 1600 rpm with butanol-pyridine-water system; amount of influx-about 0.7 ml/min: site of original spot-2.5-3.0 cm from the center ; multiple development-4 times.

Results

The Rf values and chromatograms of oligosaccharides present i n soybeans are shown in Table 1 and Figure 2, respectively.

T h e Rf values were higher in centrifugal chromatography than in ascending chromatog- raphy. Among oligosaccharides, the higher oligosaccharides showed greater differences in Rr between the centrifugal and ascending techniques.

(3)

Tech. Bull. Fac. Agr,, Kagawa Univ.

Filter paper

Table 1 Ascending and centrifugal paper chromatography of oligosaccharides (4 developments) : Rf values Sucrose Raff inose Stachyose Verbascose Sucrose Raff inose Stachyose Verbascose Sucrose Ra f f inose Stachyose Ver bascose Sucrose Raffinose Stachyose Ver bascose Ascending

chromatography Centrifugal chromatography T6y6 No 50 T6y6 No. 514 T6y6 No 51 UH

Sugar extracts from soybeans showed the presence of stachyose, raffinose, and sucrose; verbascose spots could not be identified.

The separation of monosaccharides was investigated by centrifugal chromatography, but separation of each sugar was unsuccessful under the operating conditions used for the separation of oligosaccharides

.

(4)

B u O H - C ~ H ~ N - H ~ O 1600 rpm, 0 68ml/min BuOH-AcOH-H20 1500 rpm, 0 6 8 ml/min [ 6 : 4 : 3 1 (18 5 cm Hg) 10°C

[

4 : 1 : 2 1 1 1 ° C BuOH-AcOH-H20 1500 rpm, 0 68 ml/min

[

4 : 1 : 3 1 1 O 0 C BuOH-AcOH-Hz0 1500 rpm, 0 . 6 8 ml/min

[

4 : 1 : 5 1 1 O 0 C

@ @ @ Soybean sample, A Verbascose, B Stachyose, C Raffinose, D Sucrose, Multiple development- 4 times Tbyb No 514 paper

Fig. 2 Chromatograms

Summary

Oligosaccharides such as sucrose, raffinose, and stachyose in the sugar extract from soybeans were separated clearly and detected qualitatively within a short time by use of centrifugal chromatography. The optimal operating conditions a t room temperature (about 10' in winter) were: rotation, 1500-1600rpm ; amount of influx, about 0.7 ml/min; filter paper, T6y6 No. 514; mobile phase, butanol : acetic acid : water

=4

: 1 : 5; site of original spot, 2.5-3.0 cm from the center; multiple development,

4

times.

Acknwledgments

This paper corresponds to Section 3 of Chapter

;*

(Isolation and identification of the

*

Chapter 3 of the said report consisted of the 4 following sections

1 Isolation and determination of sugars from three parts of soybeans by carbon column chromatography. Sin'itirb KAWAMURA and Minoru TADA

,

printed in this issue : Tech Bull. Fac. A g r

.

Kagawa Univ

(Kagawa Daigaku Nbgakubu Gakuzyutu Hbkoku), 18 (2), 138-41 (1967)

2. Isolation of sugars from defatted soybean meal by gel filtration Tadasi KASAI and Sin'itirG KAWAMURA, printed in the preceding issue : Ibzd , 18 ( I ) , 9-15 (1966)

3 . Separation of sugars from soybeans by centrifugal chromatography. Sin'itiro KAWAMURA, Himshi SUZUKI, and Masakazu IMAYOSI, this paper.

4. Identification of sugars by melting point and specfic rotation of free and acetylated compounds Included in the previous paper of Tadasi KASAI and Sin'itiro KAWAMURA given under 2. above.

(5)

146 Tech Bull Fac. Agr

.

Kagawa Univ sugars from soybea~ls) of the final report of research conducted under grants (grant number FG-Ja-105) authorized by Public Law 480 for the United States Department of Agriculture

.

References

(1) KAWAMUPA, S : This Bulletzn, 18,117(1967) (4) DEYL, Z

, ROSMUS, J

,

PAVLICEK, M : (2) KAWAMURA, S : Ibid

,

132 Chromatogr Revs

,

6 , 19 (1964)

(3) KAWAMURA, S

,

TADA M : Jbid

,

138

~ll+jE-fii3,

%*

%,

424-*

g E

: : k a $ & & + ~ 5 - ~ r ~ - % , ~ 7 ~ I - Z , ~ ~ ~ ~ - Z K S I L \ ~ - - - / ' . - ~ U ~ I T 3 7 4

-&?frGM-F

6 2 2 K L 9 % @ ? d T 6 $ f i : & 4 . ~ % % L ~ ~ @ ~ k & & ; s ~ ~ f i k k z's:, k %~b? D 7 k T 7 7 4

-

s&%%$&#K%h; 1 0 ° s t:

3 ,

DEB, @fi1500 - 1600; E&$IJSA%J&$J 0 7ml/%; 58fiF@fNo 514; E@#$R,

79

/ - JV : '@ @ : 7k=4 : 1 : 5 ; B , G , s & g , +~G;s>b 2 5 - 3 Ocm; ERB?!34

z

~ % # T K ~ % ? % ~ % % K ~ Z ? Y J

Lk;s\ gk

z s ~ ~ t t p z y r ~ % g s % z K ~ a

Table  1  Ascending  and  centrifugal  paper  chromatography  of  oligosaccharides  (4  developments)  :  Rf  values  Sucrose  Raff inose  Stachyose  Verbascose  Sucrose  Raff inose  Stachyose  Verbascose  Sucrose  Ra f f inose  Stachyose  Ver bascose  Suc
Fig.  2  Chromatograms

参照

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