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MICHEGM STATE UNEMERSE‘M’
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ABSTRACT

CARBOHYDRATES IN ONIONS (ALLIUM CEPA)

BY

George Menelaus Lolas

Carbohydrates make up the bulk of dry-matter of the
onion bulb. This study was made to determine qualitatively
the type of sugars present in the following varieties of
onion: Spartan Banner, Spartan Era, Granada, Ruby and
MSU (2935x2879)4535.

The techniques of paper chromatography and thin-
layer chromatography were used for the separation and identi—
fication of the sugars. Besides glucose, fructose and
sucrose nine oligosaccharides were separated by paper
chromatography.

Using n-butanol : ethyl alcohol : water = 5:3:2
by volume as solvent and multiple and overrun irrigation,
two trisaccharides, three tetrasaccharides, two pentasac-
charides and two unidentified oligosaccharides were well
separated by paper chromatography.

DeSpite the fact that these oligosaccharides are
found in very small quantities attempts were made to
determine the structures of the three tetrasaccharides

from the products of their partial hydrolysis.

George Menelaus Lolas

The Seliwanoff reaction of fructose and the
enzymatic/colorimetric determination of glucose (glucose
oxidase) were used for the estimation of the ratio of

fructose : glucose in the oligosaccharides.

CARBOHYDRATES IN ONIONS (ALLIUM CEPA)

BY

George Menelaus Lolas

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Food Science
and Human Nutrition

1972

\
A" D
A/

9‘
>6

DEDICATION

To my Father and Mother Menelaus and Anastasia

for their patience and encouragement

over the years.

ii

ACKNOWLEDGMENTS

The author is greatly indebted to his major
Professor Pericles Markakis for his guidance, encourage-
ment and patience throughout the course of this work and
during the preparation of this manuscript.

He also wishes to express his appreciation to
Professors Charles M. Stine and Grant H. Vest for their
critical evaluation of this manuscript. Special acknowledg-
ment is due to Professor Vest for providing the samples of
onions used in this investigation.

The author is most grateful to his wife Margaret
for her inspiration and encouragement throughout this

study.

iii

TABLE

DEDICATION . . . . .

ACKNOWLEDGMENTS . . .

LIST OF TABLES. . . .

LIST OF FIGURES . . .

INTRODUCTION . . . .

REVIEW OF THE LITERATURE

Methods in the Determination of

OF

CONTENTS

Methods of Chromatography .

Sugars in Onions .

MATERIALS AND METHODS .

Preparation of Sample for Analysis

Paper Chromatography.
Quantitative Analysis
Thin-Layer Chromatography

RESULTS AND DISCUSSION .

SUMMARY AND CONCLUSIONS.

BIBLIOGRAPHY . . . .

iv

Sugars.

Page
ii

iii

vi

WP

man»

12
12
l3
18
21
24
34

43

LIST OF TABLES

Page
Composition of solvent systems by volume . . . 14
Determination of fructose : glucose ratio in
onion oligosaccharides . . . . . . . . . 28
Quantitative observations on oligosaccharides
present in the onion (var. 3-Way) . . . . . 33

LIST OF FIGURES

Page
Descending paper chromatography of the onion
carbohydrates with solvent n-butanol : acetic
acid : water = 4:1:5 by vol. . . . . . . 35

Multiple descending paper chromatography of
five varieties of onions with the solvent
n-butanol : ethanol : water = 5:3:2 by vol. . 36

Multiple descending paper chromatogram of the
trisaccharides, tetrasaccharides and penta-
saccharides of the onion . . . . . . . . 37

Multiple descending paper chromatography of hy-
drolysates (complete hydrolysis) of two

varieties of onions: A, Ruby; B, 3-Way, with

the solvent n-butanol : ethanol : water =

5:3:2 by vol . . . . . . . . . . . . 38

Multiple thin-layer chromatography of three
varieties of onions: A, Ruby; B, Spartan

Banner; C, 3-Way with the solvent n-propanol :

ethyl acetate : water = 6:1:3 by vol. . . . 39

Multiple descending paper chromatography of

partial hydrolysis of tetrasaccharides iii,iv

and v of the onion with the solvent n-butanol
ethanol : water = 5:3:2 by vol. . . . . . 40

Three tetrasaccharides can be formed from
lF—B-fructosylsucrose and 6G-B-fructosylsucrose

by the substitution of the C-1 primary alco-

holic group with a fructose residue of the

sucrose molecule (transfructosidation) . . . 41

vi

INTRODUCTION

Considerable information about the onion has been
accumulated since about 1900. Some of this has given us a
new insight into disease control, adaptation, cultivar
improvement, and the commercial possibilities of manufac-
tured products.

A great deal is known about the general physiology
of the plant. Initial investigations gave the first inform-
ation about the specific effect of day-length on plants,
and showed that bulbing in the onion was dependent upon a
suitable length of day. Temperature also exerts a pro—
found influence on bulbing and flower initiation. However,
relatively little of the detailed chemistry of the plant
has been worked out. This is not surprising for, until the
development of new analytical methods, especially, of
chromatography in its various forms, searching for all but
the most obvious components of the onion was almost an
impossible task.

On the basis of nutrient content per pound of fresh
vegetable, onions ranked about midway among 31 vegetables
considered. On the nutrients per-acre basis, late onions
ranked 3 (exceeded only by mustard greens and white

potato) and early onions, 9. On the basis of nutrients

per man-hour of labor for production, late onions ranked 6
and early onions 9 or 10 (22).

Apart from these comparisons, it should be noted
that onions, compared with other fresh vegetables, are
relatively high in food energy, intermediate in protein
content, and rich in calcium and riboflavin (22).

In this study the free sugars of the onion were
identified and attempts were made to characterize some of
the oligosaccharides present.

Unfortunately, the quantities of the isolated
oligosaccharides were not large enough to allow definite
structural identification by the technique of complete

methylation.

REVIEW OF THE LITERATURE

Methods in the Determination of Sugars

 

Colorimetric tests for reducing sugars and poly-
saccharides have been known for a considerable time.
Reagents such as l-naphthol (41) for carbohydrates in
general; benzidine for pentoses and uronic acids (21);
naphthoresorcinol (46), and resorcinol disulfonic acid (34)
for ketoses are well known examples of colorimetric tests
that may be carried out in acid solution. Such tests as
these and modifications of them using aromatic amines and
phenols (6,19,47) have recently gained added importance
because of the extensive development of partition chroma-
tography for the separation and characterization of minute
amounts of sugars and their derivatives (l,6,l6,17,19,20,
44,63). Polyols and carbohydrates with a reducing group
may be detected by the Tollens silver reagent (46,68),
perhaps one of the best reagents in the art of chroma-
tography. Reducing sugars are also detectable by picric
acid (7,66), o-dinitrobenzene (16), and methylene blue,
while diazouracil is said to be specific for sucrose as
well as oligosaccharides and polysaccharides containing

the sucrose residue (51).

Volumetric procedures involving the use of potassium
ferricyanide (18), ceric sulfate (62), copper sulfate (15,
57), and sodium hypoiodite are used to determine small
amounts of reducing sugars after separation by partition
chromatography. However, experience shows that these
methods require considerable skill, are time-consuming,
and are sensitive to slight variation in the conditions.

The anthrone (12,42,43) and the 1-naphtholsu1fonate
(ll) reagents are excellent for standard sugar solutions
(42), but when applied to the analysis of sugars separated
by partition chromatography, the presence of only traces
of residual solvent developer may render them useless.
Phenol in the presence of sulfuric acid can be used for
the quantitative colorimetric microdetermination of sugars
and their methyl derivatives, oligosaccharides, and poly-
saccharides (13). The enzymatic/colorimetric determination
of glucose (glucose-oxidase) is unique for exclusively

determining glucose.

Methods of Chromatography

 

Chromatography is a separation technique of great
resolving power and considerable complexity.

Paper chromatography (5) may have had its origin
with the description by Pliny (23-79 A.D.) of the use of
papyrus impregnated with an extract of gall nuts for the

detection of ferrous sulfare, or with the studies of

"Kapillaranalyse" by Runge, Schonbein, and Goppelsroeder
in the period from 1850 to 1910.

There is little doubt that M. S. Tswett should be
given credit for discovering the principle of preferential
adsorption (adsorption chromatography) of plant pigments
on a large variety of adsorbents packed in a glass tube.

The great popularity of the present-day paper
chromatography is due to A. J. P. Martin, R. Consden,

A. H. Gordon and R. L. M. Synge (35).

Since Tswett's experiments,-chromatography has
been employed on a very small or micro scale, on a medium
scale, and on a large industrial scale. It has been uti-
lized in various geometric modifications; with one-way or
linear flow of solvent in columns, strips, sheets, or thin
layers of the sorbent (61); with two—way or transverse flow
with two different solvents in succession in sheets or
thin layers of the sorbent; and with radial flow from a
central zone or point in a sheet or layer of the sorbent
(64). It has been utilized with all kinds of liquids as
the wash medium (solution chromatography) (5,9,30,56,65).
It has been modified through the use of many different
sorptive materials such as surface-active liquids as well
as solids (9,30,64,65) (adsorption chromatography), ion
excange resins (ion—excange chromatography) (9,30), fixed
polar liquids (liquid-liquid or partition chromatography),

fixed nonpolar liquids (reversed phase partition

chromatography), paper alone (paper adsorption chroma—
tography), paper plus fixed liquids (paper partition
chromatography) (9,30,61,64,65), and polymeric gels (gel
filtration and gel permeation chromatography).

Variations of the sorptive phase have been accom-
panied by great variations of the solvents, ranging from
nonpolar hydrocarbons (sorption solvents) to polar acids,
bases, and aqueous solutions (elution solvents) (30).

With all these modifications of sorbents and solvents,
chromatography has been readily adopted to the examination
of all kinds of substances, from the smallest molecules
and ions to large colloids and biological particulates
(64,65).

Chromatography has been refined by the use of inert
gases to carry mixtures of gases or vapors through sorption
columns (gas chromatography). In these modifications, the
sorbents are usually surface active solids (gas adsorption
chromatography) or fixed, nonvolatile liquids (gas parti-
tion chromatography or gas-liquid chromatography). Gas
chromatography is useful with all kinds of gaseous and
volatile compounds.

Partridge (46,48), employing many of the methods
and solvents used in the resolution of amino acid mixtures
on paper, introduced the paper chromatography of carbo-

hydrates.

Reviewing the basic methods of paper chromatography
of sugars, Kowkabany (29) gave solvents and spray reagents
suitable for carbohydrate analysis.

Column processes are the more useful chromato-
graphic methods for the separation of pure compounds, in
quantity, from mixtures. Separation of the various solutes
from a mixture occurs in an appropriate system as a result
of differential migration of bands through a column of
stationary phase.

Mixtures of sugars (31) and their derivatives have
been separated by adsorption chromatography using a wide
variety of adsorbents, for example, magnesol, alumina,
clays (32), charcoal (69) and silicic acid. Certain of
these methods possess merits which ensure their continued
use, especially for the separation of such closely related
compounds as anomers and certain diastereoisomers. Never-
theless, these methods are subject to the unpredictability
of adsorption chromatography and are difficult, expensive,
and time-consuming.

Partition on celite columns, using both elution
and extrusion techniques, has been found to be a con-
venient and satisfactory chromatographic method for
preparative or analytical work involving carbohydrates
and their derivatives.

Another technique closely related to column

chromatography is the thin-layer chromatography. Both

involve the same types of chromatography: adsorption and
partition. The major difference lies in the location of

the stationary phase. In thin-layer (TLC) the stationary
phase is in a thin layer on a glass plate, and in column

chromatography it is in a column held in a glass tube.

Thin-layer chromatography now is considered an
indispensable tool in many laboratories, especially those
engaged in research on lipophilic natural products.

In 1938 Izmailov and Shraiber described the basic
principle underlying the process of TLC, and they used the
method for separating plant extracts.

Martin and Synge (36,37) develOped a method of
partition chromatography for separating amino acids and
their derivatives.

Consden, Gordon and Martin (10) in 1944 started
using filter paper, in order to carry out partition
chromatography on a microscale.

Kirchner (27) in 1950 attempted to separate
lipOphilic mixtures using adsorption chromatography on
impregnated filter paper, and later glass fiber paper
coated with silicic acid or alumina.

Kirchner and Miller (28,38,39) proceeded to
investigate methods of separating terpene derivatives
on thin adsorbent plates.

It is astonishing that the method of thin-layer

chromatography remained in obscurity until 1956 when

Stahl described equipment and procedures for the prepara-
tion of chromatoplates and demonstrated the potential use-
fulness of TLC in the fractionation of substances other
than terpenes.

Pastuska (49) described a procedure for the quanti-
tative titrimetric determination of sugars in zones scraped
off thin-layer chromatograms on kieselguhr G. The method
is based on a wet combustion with potassium dichromate-

sulphuric acid and titration of the unused dichromate.

Sugars in Onions

 

Sugar content of onion bulbs has been measured in
numerous physiological studies (22). Bennett (4) in 1941,
reporting two year's work, measured reducing sugars (as
glucose) and non-reducing sugars (as sucrose) of
"Ebenezer" onions when they were placed in storage and
after 3 1/2 months of storage. In the first year the
bulbs contained, on a dry—weight basis at the beginning
of storage, 60 per cent of non-reducing sugars and 5.2 per
cent of reducing sugars; in the second year, the contents
were 42 per cent and 17 per cent, respectively. The
percentage loss of total sugars varied from 3.0 to 8.1
per cent, depending on storage conditions. However, reduc-
ing sugars increased in all storages, especially at low
temperatures (33 to 34 F), where they rose from 5.2 to 21

per cent the first year and from 17 to 29 per cent the

10

second year. Karmarkar and Joshi (24) in 1941 also found
that reducing sugars increased during low-temperatures
storage, and this was again observed by Yamaguchi et a1.
(70) (1957), working on "Southport White Globe." These
latter workers also found, contrary to Bennett's observa-
tion, that reducing sugars exceeded non-reducing sugars
at the start of storage.

Some investigators (50,55) have reported arabinose,
xylose, ribose, rhamnose, lactose and raffinose in the
onion bulb, but most workers say that the onion bulb con-
tains only glucose, fructose, sucrose and higher oligo-
fructosides (2,3,8,l4,33,40,58,67).

Bacon (2,3) found glucose, fructose, sucrose, and
a series of water-soluble, mainly non-reducing oligo-
saccharides whose chain length did not exceed eight
residues. No fructose polysaccharide was found. The
oligosaccharides are distributed unevenly in the mature
onion bulbs, being absent from the outer scales, while in
the inner ones they may constitute more than half the
soluble carbohydrate. The oligosaccharide is concentrated
more at the base of the scales than at the top and con—
siderable variations occur from point to point round the
circumference of each scale. Bacon (3) identified two
trisaccharides, fructose : glucose ratio 2:1, as 1F-8-

fructosylsucrose and 6G-B-fructosylsucrose. These were

11

followed by the first tetrasaccharide, fructose : glucose
ratio 3:1.

In the onion, where no polysaccharide fraction is
found, there can be no doubt that the oligosaccharides
are present as such in the intact bulbs, and it is tempting
to argue that the similarity in the trisaccharide fractions
of other Monocotyledon species shows that they occur as
a result of normal physiological processes.

Sugars of onions and other Monocotyledons have
been studied extensively, in an attempt to find the

mechanism of fructosan biosyntehsis in plants (53,54).

MATERIALS AND METHODS

Preparation of Sample for Analysis

The onions for this study were commercial onions
from the fields around East Lansing, Michigan. They were
planted in April, 1971 and harvested in September, 1971.
Maleic hydrazide had been used as pre-harvest treatment
to prevent sprouting. After harvesting they were stored
in a refrigerator at 0-3°C. The following varieties were
investigated: Spartan Banner, Spartan Era, Granada, Ruby
and MSU (2935 x 2879)4535 (3-way).

In the first experiments two onion bulbs of each
genotype were cleaned from the outside dry material,
divided into representative pieces and one hundred grams
were weighed out (representative pieces of bulb), chopped
into smaller pieces, covered with 80% hot ethyl alcohol and
heated on a steam bath for one hour. The solution was
decanted and the onion pieces were comminuted into a Waring
blender with 80% ethyl alcohol. The comminuted material
from blender was again heated for half an hour on a steam
bath.

The mixture was filtered through a Buchner funnel
and the filtrate, combined with the previous decanted
solution, was transferred to a Buchler flash evaporator
and concentrated to about 100 ml at 38°C. The concentrate

12

13

was centrifuged at 10,000 r.p.m. for 15-20 minutes. The
clear syrup was stored at 0°F to be used later for chroma-
tographic work.

No other clarification or preparation was necessary.

In later experiments in which there was an interest
in securing greater quantities of oligofructosides which
normally are found in the center of the bulb the following
procedure was employed.

The four outer scales of the bulbs were discarded
and the remaining scales, one kilogram in all, underwent
the same treatment as before. A concentrated extract

syrup of 200 ml was obtained.

Paper Chromatography

 

Separation by paper chromatography is based upon
differences in the partition coefficients of the sugars.
Separation of optical enantiomorphs such as DL-sugars is
not possible by paper chromatography. The process involves
countercurrent partition between a stationary cellulose-
water complex, filter paper which contains 20% water and
a great number of fibers, and a mobile phase composed of
an organic solvent, or mixtures of solvents, containing
some quantity of water.

The choice of solvent is very important and particu-
larly the water content is a critical factor, since the

solubility of a sugar in the mobile phase will define its

14

rate of movement on the paper chromatogram and thus affect
the degree of separation of a mixture of sugars.

The technique of descending chromatographic pro-
cedure was employed using Whatman No. l chromatographic
paper. The onion syrup was applied on a line 2.5 inches
from the top of the paper. Known sugars were also applied
at one inch intervals across the paper. The standards had
a concentration of 1% and were prepared in 10 per cent
aqueous iso-prOpyl alcohol and stored in the refrigerator.
After application the spots were dried with a cold air cur-
rent from a hair drier. A micropipette was used to apply
the samples.

A number of solvent systems were used in an effort
to separate as many of the sugars as possible. Table 1

shows the composition of the solvent systems tested.

TABLE l.--Composition of solvent systems by volume.

 

1. n-Butanol : acetic acid : water = 4:1:5
2. n-Butanol : ammonia : water = 4:1:5
3. n—Butanol : ethyl acetate : water = 4:1:5
4. n-Butanol : acetic acid : ethanol : water = 8:1:1:10
5. n-Butanol : ethyl alcohol : water = 5:3:2
6. n-Butanol : iso-propyl alcohol : water = 3:5:2
7. n—Butanol : pyridine : water = 6:4:3
8. Benzene : acetic acid : methanol = 1:1:3
9. n-Propyl alcohol : ethyl acetate : water = 6:1:3
10. n-Propyl alcohol : water = 7:1
11. iso-Propyl alcohol : water = 4:1
12. iso-Propyl alcohol : acetic acid : methanol = 7:2:1

 

15

The solvent system which showed the best resolution
was #5, n-butanol : ethyl alcohol : water = 5:3:2, by
volume.

This solvent system is monophasic. Generally,
monophasic solvent systems are considered superior of
biphasic solvent systems because the composition of the
biphasic solvent mixtures renders them temperature-
dependent and a constant-temperature room for chromatography
and solvent equilibration should, therefore, be used to
avoid separation of the solvent mixture into two phases
during development.

MonOphasic systems generally consist of water, a
water-soluble organic solvent and an organic solvent which
is immiscible with water. The amount of the water-soluble
organic solvent (ethyl alcohol in the present case) deter-
mines the amount of water which may be added to the mixture
if it is to remain monophasic and this is important,
because it is the water content which in practice controls
the mobility of the carbohydrates.

The techniques of multiple and overrun development
were used for the separation of onion carbohydrates.

Multiple chromatography includes procedures in
which development is repeated either in the same direction
as in the first run (multiple development) or in a direction
perpendicular to the flow of the first system (two-

dimensional chromatography). This can be done by using the

16

same solvent or a different kind of solvent system. In
the present case the same solvent system was used (except
in a few cases where two different solvent systems were
used) and always in the same direction.

Overrun development is employed when the RF values
of the components of the mixture are too low. In such
cases, especially where the mixture does not contain com-
ponents with high RF values, it is possible to allow the
solvent front to run off the paper (overrun development).
In this technique, the development does not end when the
front of the solvent system reaches the lower edge of the
chromatogram, but it is allowed to continue with the solvent
freely dripping from the paper. In order to prevent any
irregularities in the flow of the solvent, the lower edge
of the descending chromatogram was cut in saw-tooth fashion
(each tooth about one inch wide and one inch long).

The papers and the chamber were saturated with
solvent placed in two large plates on the bottom of the
chamber for about four hours prior to the introduction of
the rest of the solvent to the troughs of the chamber for
the irrigation to start.

The development was continued for 48 hours, and for
the separation of higher oligosaccharides the following
technique was employed. The chromatograms were removed
from the chamber, dried in the chromatographic oven at

40°C and were put again into the chromatOgraphic chamber

17

for a new development in exactly the same way as before.
This was repeated a third time, so that, the whole procedure
took about seven days.

After drying, the papers were sprayed with the
spray reagent and heated to 105°C for ten minutes or until
colored spots appeared. The spray most often used was the

benzidine spray (56). This was made up of:

a. Benzidine, l g in glacial acetic acid 40 ml

Trichloroacetic acid . . . . . . . 30 g 1 vol.
Water . . . . . . . . . . . . 40 ml
b. Acetone 9 vol.

The benzidine is dissolved in the acetic acid, the
trichloroacetic acid is dissolved in the water and the two
solutions are then mixed. The resultant monophasic solu-
tion is stable in the refrigerator although it slowly
darkens in color; it is diluted with acetone immediately
before use.

Spot-localization is either based on the reducing
properties of sugars and sugar alcohols (alkaline silver
oxide, periodic acid, dinitrosalicylate), or on the conden-
sation of acid degradation products of sugars with either
a reactive phenol (naphthoresorcinol or phloroglucinol),
or an aromatic amine (aniline, diphenylamine, benzidine or
di-anisidine) to give colored derivatives.

Other spot-localization reagents tried were the

naphthoresorcinol reagent, specific for ketoses (46), the

18

ammoniacal silver nitrate solution for reducing sugars (48)
and the periodate-permanganate reagent useful for the detec-

tion of reducing substances (31).

Quantitative Analysis

 

In order to quantify the sugars by paper chroma-
tography the following procedure was used.

Two lines were drawn lengthwide 2.5 inches from
the edge of the paper. Two more lines were drawn 2 inches
and 2.25 inches from the top. The application of the
sample was made by transverse streaking in the zone between
2 and 2.25 inches from the top. The chromatograms were
developed as in the qualitative analysis. A blank paper
was also developed with the same way to serve as a control.
Then two strips containing the unknown sugars, sucrose, and
stachyose, used as location agents since stachyose cor-
responds with the fourth oligosaccharide following sucrose
and sucrose is followed by the first oligosaccharide, were
cut from the paper and sprayed with the benzidine spray to
locate the distance that the sugars had travelled.

The strips were then matched with the center portion
and the center was cut into sections corresponding to the
location of the sugars. Each section was again cut into
small pieces about one square centimeter and transferred
to 250 ml beakers. Enough deionized water was added to
cover the paper clippings. The beakers were covered and

stored overnight in the refrigerator. During this time

19

the sugars became equally distributed in the free liquid
and that between the cellulose fibers. The eluate was then
passed carefully through a fine sintered glass filter to
retain the cellulose lint of the paper.

The filtrates were concentrated by a Buchler flash
evaporator (vacuum) to 5 ml at 40°C. These were hydrolysed
by dilute hydrochloric acid at 70°C for two hours in a water
bath. After hydrolysis, the solutions were neutralized by
dilute sodium hydroxide and the necessary dilutions were
made for the determination of glucose and fructose in order
to find the ratio of fructose : glucose in the oligofructo-
sides.

The reason sintered glass filter was used is that
if cellulose fibers are present in the solution of oligo-
saccharides these could be hydrolysed to glucose and a
considerable error would be introduced in the determination
of glucose. The purpose of the blank paper was to check
the presence of cellulose in the filtrate and the influence
of solvents in the determination of both the glucose and
fructose.

The enzymatic/colorimetric determination of glucose
(25,26) was used, according to SIGMA Technical Bulletin
No. 510 published by SIGMA chemical Company (3500 De Kalb
St., St. Louis, Mo. 63118, U.S.A). In each tube of blank,
standard and unknown solutions were added in the order indi-

cated, 0.1 m1 of color reagent solution (50 mg of

20

o-dianisidine dihydrochloride, Stock No. 510-50 plus 20 ml
water), 5.0 ml enzyme solution (contents of one PGO enzymes
capsule plus 100 ml water in amber bottle). To the blank
was added 0.5 ml water, to the standard 0.5 m1 of a 20—fold
dilution of glucose standard solution, concentration

1 mg/ml, Stock No. 635—100 (1 part solution and 19 parts
water) and to the tubes of the unknown solutions 0.5 of
each unknown solution, respectively. The tubes were mixed
well, incubated at 37°C for 30 (:5) minutes, and at the end
of this period, the tubes were removed from the water bath
and the absorbance of standard and unknown solutions, using
the blank as reference, was read at 450 mu (:25mu). The
reading of the brown color was completed within an addi-
tional 30 minutes that the method required.

According to the SIGMA bulletin no calibration
curve is required because all instruments with which they
have prepared a calibration curve produced practically a
straight line (Beer's law). To verify the above statement
of the Company two solutions of glucose containing 25y
and 50y per ml were used and their absorbance was measured.
The absorbance of the second glucose solution was found to
be almost exactly twice that of the first one (0.230
against 0.117).

For the determination of fructose the reaction with

resorcinol and hydrochloric acid (52) was employed.

21

To 1 ml of a solution containing 10‘50Y of fructose
was added 4 ml of a reagent prepared by mixing 7 parts of
30% hydrochloric acid (5 volumes of conc. hydrochloric acid
plus 1 volume of water) with 1 part of a reagent prepared
by dissolving 0.1 g of resorcinol and 0.25 g of thiourea in
100 ml of glacial acetic acid. The reaction mixture was
heated 10 minutes in a water bath at 80°C with frequent
shaking and then cooled in running water. The appeared
purple color is measured at 515 mu.

Lowering the temperature of the water bath from
100°C to 80°C reduced somewhat the intensity of color
produced but increased the specificity of the reaction.
Thus glucose had a negligible effect at a concentration of
1 mg/ml.

Absorbance for both glucose and fructose was

measured with a Beckman D U SpectrOphotometer.

Thin—Layer Chromatography

Many attempts to separate sugar mixtures by thin-
layer chromatography have been made since Stahl's (59,60)
first experiments. However, results obtained with all
known adsorbent mixtures have proved unsatisfactory (49),
and identification of many of the most important sugars
was highly dubious.

Ovodov gt_al, (45) have stressed that poor separa-

tion of some of the more common sugars and the low

22

capacity of the chromatoplates are responsible for the lack
of general acceptance of thin-layer chromatography in
research work on carbohydrates.

Pre-coated plates were mostly used. It is generally
considered that pre-coated plates surpass those made in the
lab in strength during multiple development employed for
the increase of the separating power or for the purification
of the layer prior to certain quantitative assays.

Despite the difficulties of separation of carbo-
hydrates on thin-layer chromatograms the following materials
were used.

Pre-coated TLC plates (5X20 cm) with silica gel
F-254, layer thickness 0.25 mm distributed by Brinkmann
Instruments, Inc. (Westbury, N.Y. 11590). The plates were
used after impregnation with 0.1 N boric acid or in the
state they were obtained (49).

Pre-coated polyamide F-254 (20x20 cm) plates pur-
chased from the same company, and uniplate pre—coated TLC
plates with Avicel, thickness 0.25 mm of Analtech, Inc.

(100 South Justison St., Wilmington, Del. 19801) were also
used. The latter one was impregnated or not with 33 mM

of KZHPO4 (23) and was used more frequently since it gave

the best results, especially after the impregnation with

K HPO . The plates, after impregnation, were dried for 24

2 4

hours at room temperature.

23

Solvents used were some of those employed for paper
chromatography but the best results were obtained by the

solvent systems: n-butanol : ethyl alcohol : water =

5:3:2, and n-propyl alcohol : ethyl acetate : water
6:1:3 by volume.

The cabinet was saturated with the solvent and then
the plates were introduced. The chromatograms were routinely
developed three times in the same solvent (multiple thin-
layer chromatography) until the solvent front was about
1 l/2 inches from the top. The plates were dried with warm
air before the next development.

The Spray reagent of benzidine was used for the
development of the spots. The presence of sugars was indi-
cated by the appearance of dark brown spots (except the
fructose spot which was yellow) on a light yellow-brown
background. The identification was made as in paper

chromatOgraphy.

RESULTS AND DISCUSSION

Paper chromatographic studies of onion extracts
from all five varieties showed the presence of glucose,
fructose, sucrose, two trisaccharides, lF-B-fructosylsucrose
and 6G-B-fructosylsucrose, three tetrasaccharides and two
or probably four pentasacchflrides.

Figure 1 shows that in onion only glucose, fructose,
sucrose and higher oligosaccharides occur. When the
chromatogram was irrigated for about 24 hours the RF values
of xylose, rhamnose, ribose and arabinose are higher than
the RF values of the sugars present in the onion. It should
be noticed that only one trisaccharide has been separated.
If multiple and overrun chromatography is applied more
oligosaccharides will be separated, and the differences in
the partition coefficients of xylose, rhamnose, ribose and
arabinose will be much greater than those of the sugars
of the onion. These results are in conflict with the
results of some other workers (50,55) who reported that
they found these sugars in the onion bulb.

Figure 2 shows that the five onion varieties
examined had the same sugars. The chromatogram was irri-
gated twice, each time for 48 hours, and the solvent was

allowed to run off the paper. In addition to glucose,

24

25

fructose and sucrose, six oligosaccharides were separated.
When the chromatogram was irrigated for three 48 hour
periods,glucose, fructose and sucrose ran off the paper and
seven to nine oligosaccharides separate as it is shown in
Figure 3. The oligosaccharides are named here for simplicity
i,ii,iii, etc. starting with the oligosaccharide which
follows the sucrose. The trisaccharide i is the lF-B-
fructosylsucrose and the trisaccharide ii the 6G-B-fructosyl-
sucrose, according to Bacon (3). It is to be noticed that
stachyose follows the oligofructoside iv in all its movement
even after the three successive irrigations. The solvent
used was n—butanol : ethyl alcohol : water = 5:3:2 whereas
that of chromatogram of Figure l was n-butanol : acetic
acid : water = 4:1:5. Multiple and overrun chromatography
with the latter solvent system did not give equally good
results even when continued for longer time. Perhaps,
this is due to the biphasic system involved, the composition
of which is susceptible to even small changes of tempera-
ture. It is also possible that some esterification of
butanol with the acetic acid may occur upon long standing.
The n-butanol : ethyl alcohol : water = 5:3:2 by vol. solvent
system as monophonic is not subject to similar changes.

The chromatogram shown as Figure 4 has been obtained
after complete hydrolysis of all the carbohydrates present
in the onion bulb. It shows that only glucose and fructose

are present in the hydrolysates. These results indicate

26

that galactose, raffinose and lactose are not present in
onions as reported by other investigators (50) since, in
that case, after complete hydrolysis, galactose would be
present. Also, stachyose cannot be considered to be present
in onions despite the clear correspondence with the fourth
spot that the onion samples give, because galactose is a
part of the stachyose molecule.

Figure 5 shows a thin—layer chromatogram on Avicel.
Avicel, in combination with n-propyl alcohol :ethyl acetate
water = 6:1:3 as a solvent, provided the best separation of
all sorbents and solvents tried in the thin-layer chroma-
tography. With the help of thin—layer chromatography no
more than five oligosaccharides could be separated. This,
again, shows the superiority of paper chromatography upon
the thin—layer chromatography of carbohydrates. Again the
raffinose spot did not correspond to any of the Spots of
onion sugars and it was located between the spots of
lF-B—fructosylsucrose and 6G-8—fructosylsucrose. The plate
was developed with the same solvent system three times,
each development taking about three hours. The plates were
dried with warm air current from a hair drier between each
development.

The paper chromatogram of Figure 6 shows the pro-
ducts of partial hydrolysis of tetrasaccharides iii,iV
and v. The partial hydrolysis was carried out with 0.005 N

hydrochloric acid at 80°C for 30 minutes in a water bath

27

and after the end of this period the solution was neu—
tralised with 0.005 N sodium hydroxide.

The quantitative determination was done as described
previously for the estimation of fructose : glucose ratio.
The results, an average of two determinations, are shown
in Table 2.

No standard curve was prepared for the determination
of fructose, but standard fructose solutions containing 20
and 40y/ml were included in each set of estimation; these
gave absorbancies of 0.168 and 0.340, respectively.

Data in Table 2 show that oligosaccharides iii,iv
and v are tetrasaccharides and the vi and vii pentasac-
charides. Using the ammoniacal silver nitrate spray all
the oligofructosides were found to be non—reducing. The
products of partial hydrolysis of tetrasaccharides iii,iv
and v (Figure 6) all gave fructose, sucrose and trisac—
charides i and/or ii; v is the only one which gave glucose.

In 1950 Blanchard & Albon, and Bacon & Edelman
simultaneously found that trisaccharides with a fructose
glucose ration of 2:1 were formed during the hydrolysis of
sucrose by ivertases. In the same year a decisive simpli—
fication of the adsorption chromatography of sugars on
activated carbon was introduced by Whistler and Durso (69),
and by its use three trisaccharides, 6F-B-fructosylsucrose,
lF-B-fructosylsucrose and 6G-B—fructosylsucrose, were

isolated and characterized; the first two are now known in

28

 

 

na.v 0.0m Hm xo.OH o.oH mm.o mmo.o o.NH "on.ma o.mH vN.o nmo.o HH>
ma.v N.mm Hm Xm.mv m.mv vN.H mmv.o o.vm "axo.wm o.¢m mw.o mHH.o fl>
mH.m o.moaum xm.mm m.mm mm.o Hmm.o o.mm “on.mm o.mm vo.a vwm.o >
oa.m o.omHnm xo.mm o.mm om.o mnm.o m.am "me.am m.Hm mo.H ovm.o >H
ma.m o.ommHomXo.mm 0.0m mo.o Hmm.o o.mmHnmxo.mm o.mm om.o va.o HHH
0H.N o.vomuoaxv.om w.om mN.H mmv.o o.ovmumxo.m¢ o.me mm.o «mm.o HA
mm.a 0.0mmuomxo.mm o.mm on.o mmm.o m.mmmnme.mm m.om MH.H vmm.o A
II I: o.ov ll ovm.o It 0.0m In vmm.o .pum
Hs\> Hs\>
.Ewowwp 030 Momma Eouw momma Eouw
mo mmmum>m pomuuxo wo .HE\> HODOMM .E: mam uomuuxo mm “HE\> HOpomw .81 omv
Oaumu coflusaflp Mom ocoo QHOmQ< COADSHHU H m ocoo QHOmQ< mHmEmm
. .uomnuoo .uomuuoo
mmousHo . .ocoo .ocoo

"mmouosum

 

COHpmsHEHmpmp mmODosum sowpmcaeuwump mmoosao

V P F p 7 F ? VII)? i

.mmpflumnoommomflao GOHco SH oflpmu mmoosam u mmouosum mo COMDMCHEHODOQII.N mqméa

29

the crystalline form. So far, no other trisaccharide of
this general type has been identified. It will be noticed
that these are the only three trisaccharides that can be
formed by substitution of a primary alcoholic group of
sucrose, and all three have been found in plant tissues.
Oligofructosides, in general, are presumed to be derived
from one or the other of these trisaccharides by addition
of further B-fructofuranosyl residues.

Since in the onions tested 6F-B-fructosylsucrose
was absent it may be assumed that some enzyme is responsible
for the transfructosidation of C-1 of the fructose residue
of sucrose.

Schlubach and Koehn (53), trying to explain the
presence of lF-, 6G-B-fructosylsucrose and bifurcose (a
tetrasaccharide) in rye, suggested that the simplest expla—
nation is to accept the existence of two different enzymes,
one which acts on the 1- and the other on the 6- position
of the fructose residue of the sucrose molecule.

Since the onion tetrasaccharides gave among the pro-
ducts of their partial hydrolysis 1F- and 6G-8-fructosyl-
sucrose, and since they are non-reducing it could be
assumed that they are products of further transfructosida—
tion (transfer of fructose from the molecule of sucrose
with the help of enzyme and substitution of the primary

alcoholic group of fructose residue of trisaccharide of

C-l). It should be also taken into consideration that no

30

reducing compounds could be detected in the products of
partial hydrolysis except glucose and fructose. Three
tetrasaccharides would therefore be possible through trans-
fructosidation of the two trisaccharides, as it is shown in
Figure 7.

At least, tentatively, the structures III,IV and V
may be given to the three tetrasaccharides iii, iv and v,
respectively. The basis of the given structures is that
only tetrasaccharide v gives glucose during partial
hydrolysis and the spots corresponding to sucrose and
fructose are much more intense than in the case of the
other two. Also, this is the only one which has no sub-
stitution of C-6 of the glucose, thus differing from the
other two. Knowing that the C-6 bond of sugars is
hydrolysed with difficulty, it becomes understandable
why glucose is absent in the products of partial hydrolysis
of tetrasaccharides iii and iv.

Structure III is justified on the basis of the
partial hydrolysis products, lF-B- and 6G-B-fructosylsucrose
which give spots stronger than those derived from the other
two tetrasaccharides. In addition the fructose spot of
the partial hydrolysate of iii is weaker than that of the
other two tetrasaccharides.

Structure IV given to tetrasaccharide iv can be
justified as follows. iv, hydrolysed, can give the

trisaccharide ii and another trisaccharide which differs

31

from i in that the bond between the glucose and fructose
is not between C-1 and C—2 but between C-1 and C-6 of
glucose. If this new trisaccharide has the same partition
coefficient with i then its spot is understandable on the
chromatogram. If this new trisaccharide does not really
exist then product i could be derived from iv isomerized
to iii during acid hydrolysis. Indeed tetrasaccharide iv
upon partial hydrolysis gives a spot corresponding to
tetrasaccharide iii.

Although the situation in the two previous cases
looks relatively easy to explain, that of tetrasaccharide v
is more complicated. The spots corresponding to fructose,
glucose, sucrose and trisaccharide i are easy to explain,
but not those corresponding to trisaccharide ii, tetra—
saccharides iii, iv and the spots in traces between sucrose
and i, and i and ii. Glucose here is at the end of the
molecule and isomerization by partial breaking and rebinding
cannot occur in such a way so that non-reducing products
result.

The only remedy in this situation would be to
secure greater quantities of these tetrasaccharides so
that the methylation technique can work to give the solu—
tion to the real structures.

If further transfructosidation is proved to be
correct then these tetrasaccharides can give four different

pentasaccharides. Thus each one could give:

32

F2-1F2-1G6-2F1-2F (A)

(iii) F2-lF2-lG6-2F
F2-lF2-lF2-lG6-2F (B)
F2-lG6-2F1-2Fl—2F (C)

(iv) F2—lG6-2Fl-2F
F2-lF2-1G6-2Fl-2F (D)
(v) F2—1F2—1F2-1G F2-1F2-1F2—lF2-1G (E)

From the possible five pentasaccharides A and D
are identical; therefore only four different pentasac-
charides are possible. Two oligosaccharides of the onion
had a fructose : glucose ratio 4:1 and are assumed to be
pentasaccharides. The other two oligosaccharides appearing
when the irrigation is continued for longer time are
possibly also pentasaccharides.

Some rough estimates of the quantities of these
oligosaccharides based on glucose determination and the
quantity of the onion sample applied on paper are shown
in Table 3.

From the results in Table 3 one can visualize how
difficult it is to deal with these oligosaccharides. Thus,
more sensitive techniques of detection should be introduced
in every step of the research. Possibly, some kind of
column chromatography with the proper adsorbent material
and solvent system could be used for the separation of

greater quantities of these oligosaccharides.

33

TABLE 3.--Quantitative observations on oligosaccharides
present in the onion (var. 3-Way).

 

Yield in mg/100 g fresh wt.

 

oligofructoside of tissue analyzed
1 186
ii 80
iii 35
iv 11
V 7
vi 3

vii 1

 

SUMMARY AND CONCLUSIONS

The carbohydrate constituents of five varieties of
onions examined were found to be fructose, glucose, sucrose,
two trisaccharides, lF-B-fructosylsucrose and 6G-B-fructo-
sylsucrose, three tetrasaccharides and at least two
pentasaccharides. Paper chromatography using as solvent
n—butanol : ethyl alcohol : water = 5:3:2 by volume has
proved to be an excellent method for the separation of
minute quantities of sugars present.

Further research is needed for the verification of
structures proposed for the tetrasaccharides and penta-

saccharides present in the onion bulb.

34

 

start line

A A ‘

 

Suc .0 O

(31.0 0

no

solvent front

__

 

A W —v—‘
w
v

v

 

 

 

Figure l.--Descending paper chromatography of the

onion carbohydrates with solvent n-butanol
water 4:1:5 by vol. Gl., glucose; Fr.,
sucrose; Xyl., xylose; Rh., rhamnose; Rib.
arabinose; i, lF-B-fructosylsucrose.
B, Spartan Era.

Varieties:

acetic acid

fructose; Suc.,
, ribose; Arab.,
A, Ruby;

36

 

start line

 

A‘. F} 13")
e - xii; -A, - - A 4
c3
dStach

Suc.é:}

Q
CDC? C> C33 0 a ooeiisw
C) O 0 CE Q 8. 000
D C) C3 O D @0003}:
Q Q Q Q C3 0 can;

 

 

 

Figure 2.-—Multip1e descending paper chromato-
graphy of five varieties of onions with the solvent
n-butanol : ethanol : water = 5:3:2 by vol. Gl., glucose;
Fr., fructose; Suc., sucrose; Stach., stachyose; i,ii,
trisaccharides; iii,iv,v, tetrasaccharides; vi, penta-
saccharide. Varieties: A, Ruby; B, Spartan Era; C,
3-Way; D, Spartan Banner; E, Granada.

ix
viii
vii .
v1

iv

iii

ii

 

! -1-

 

nw—- ._.__ __.__.-_.___ .7.___._ »._._———. —-

Figure 3.--Multiple descending paper chromatogram
of the trisaccharides, tetrasaccharides and pentasac-
charides of the onion. Variety: 3-Way. From below,
bands i and ii are trisaccharides, bands iii,iv and v
tetrasaccharides, vi and vii pentasaccharides and viii and
ix unidentified.

 

 
   
  

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. 4310.94.01.42. H. -. ... .. ... . u“ .. . .
ru. w.. . . . ...Ga.w.l.0." ...II .. ...; ‘3’. .n .0 .. 9. $ fl
9‘ . 0 I . v .....Ivfittr. .-. an. ‘9‘ ... .L. t..' 9 . p
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38

 

start line

03>
0w

 

.0620

Figure 4.--Multiple descending paper chromatography
of hydrolysates (complete hydrolysis) of two varieties of
onions: A, Ruby; B, 3-Way, with the solvent n-butanol :
ethanol : water = 5:3:2 by vol. Gl., glucose; Gal.,
galactose; Fr., fructose.

 

 

 

 

start line

 

 

 

Figure 5.--Multiple thin-layer chromatography of
three varieties of onions: A, Ruby; B, Spartan Banner;
C, 3-Way with the solvent n-propanol : ethyl acetate °

water = 6:1:3 by vol. Gl., glucose; Fr., fructose; Gal.,
galactose; i,ii, trisaccharides; iii,iv,v, tetrasaccharides.

4O

 

iii iv v

 

start line B-Way partially hydrolysedA
v C3 '

1:18 0 8 E?
19 C’Q GU

SucQ D (3 O a

“go $008

 

 

 

Figure 6.--Multiple descending paper chromatography
of partial hydrolysis of tetrasaccharides iii,iv and v of
the onion with the solvent n—butanol : ethanol : water =
5:3:2 by vol. Gl., glucose; Fr., fructose; Suc., sucrose;
i,ii, trisaccharides; iii,iv,v, tetrasaccharides; vi, penta-
saccharide. The unhydrolysed sugars of 3-Way are shown
for a comparison.

 

 

 

 

 

 

 

III
1. 1 1 6 a 6% 6 6 .
F F G F
21 2 2 '
l 1 1
II
2 2
6‘L 6 6 1"
F G F
G 6 6‘L 6.
6 -B-fructosylsucrose F G F F

Figure 7.-—Three tetrasaccharides can be formed from
lF-B—fructosylsucrose and GG-B-fructosylsucrose by the
substitution of the C-1 primary alcoholic group with a
fructose residue of the sucrose molecule (transfructosida—
tion); F, fructose; G, glucose.

BIBLIOGRAPHY

42

10.

ll.

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