ANALYTICAL BIOCHEMISTBY 24, 452-461 (1968)

Separation and Identification of Monosaccharides from

Biological Materials by Thin-Layer Chromatography
ANDREW E. GAL
Laboratory of Neurochemistry, National Institute of Neurological Diseases
and Blindness, National Institutes of Health, Bethesda, Maryland 20014

Received October 20, 1967

The qualitative and quantitative determination of the monosaccharides

obtained by enzymic or chemical hydrolysis of glycolipids and glyco-
proteins is essential for elucidating the structure of these materials. The
products which originate from the glycosidic portion of these molecules
are: neutral sugars, amino sugars, acylamino sugars, and neuraminic
acids. These monosaccharides have been separated and purified by a
combination of ion-exchange chromatography, paper or gas-liquid chro-
matographic separations (1, 2). After these separation procedures, quan-
titative determinations have been carried out by calorimetric assays.
In the present work, we sought to replace the paper chromatographic
techniques by more convenient thin-layer chromatography procedures.
Kliser and Masera first indicated the possibility of separating monosac-
charides by thin-layer chromatography (3). A great variety of adsorb-
ents and diverse impregnation techniques have been examined for
neutral sugars (4, 5). The separation of amino sugars on cellulose-
coated plates was described by Giinther and Schweiger (6). The separa-
tion of neuraminic acids was described by Granzer (7). More recently
Meindl and Tuppy (8) and Klenk .et al. (9) used this technique for the
separation of neuraminic acid derivatives.
The present work describes systems which reliably separate the mono-
saccharides characteristically arising from the hydrolysis of naturally
occurring hexose and neuraminic acid-containing materials. Among the
accomplishments in the course of this work was the development of a
facile procedure for the separation of glucose from galactose. Silica gel
G plates were employed without modification in our experiments. The
impregnation of thin-layer plates with inorganic salts employed by many
workers in this field did not improve the separation on silica gel G plates
in our solvent systems.
452
 THIN-LAYDR CHROMATOGRAPHY OF MONCSACCHARfDES 453

MATERIALS
D-Glucose was obtained from Mallinckrodt Chemical Co. and was re-
crystallized twice from water/methanol (l/l v/v), 1 ml/gm. n-Galactose
was obtained from Nutritional Biochemical Corp. and was recrystallized
twice from water/ethanol (l/3 v/v), 3 ml/gm. n-Galactosamine hydro-
chloride, n-glucosamine hydrochloride, N-acetyl-n-galactosamine, and N-
acetyl-n-glucosamine were obtained from the same source. N-Acetyl-
neuraminic acid (synthetic) and N-glycolylneuraminic acid (95%) were
obtained from Sigma Chemical Co. Human erythrocyte globoside was a
gift from Dr. Tamio Yamakawa, .University of Tokyo.
Silica gel G was a Merck A. G. product. The solvents were U.S.P.
standard quality. The reagents used for the spray solutions were ana-
lytical-grade materials.

METHOD
Thin-Layer Chromatography. The silica gel G plates were prepared
according to Stahl (10). A Shandon Unoplan leveler was used. The
aperture of the gel spreader was adjusted to 250 p. After drying the
plates for 1 hr in air, they were heated for 30 min at 110°C and kept
subsequently in a desiccator over Drierite. From 0.5 to 30 pg portions
of the sugars were spotted at 2 cm intervals from the edge of the plate.
The developing solvent was allowed to ascend to about 1 cm from the
upper rim. The drying was accelerated by a stream of warm air.
Sodwent Systems. In the present experiments, the following solvent
combination were found to provide satisfactory resolution of monosac-
charides: (A) n-propanol/water (7/l v/v) ; (B) n-propanol/water (7/1.5
v/v) ; (C) n-propanol/ethyl acetate/water (5/l/4 v/v/v) ; (D) methyl
acetate/isopropanol/water (18/1/l v/v/v) ; (E) methanol/water (5/2
v/v).
Visualization. The following spray reagents gave the best results:
(1) Silver nitrate. The reagents described by Trevelyan et al. (11)
were used with the following modifications: A solution of 3 gm of silver
nitrate in 12 ml of water was added to 500 ml of acetone. The plates were
sprayed with this solution and dried, and then the spots visualized with
a 0.5 N ethanolic (95%) sodium hydroxide solution (50 ml of 10 N
sodium hydroxide in 450 ml of absolute ethyl alcohol). The spraying
with thiosulfate solution was not feasible and was omitted. The stains
were very intense and stayed distinct for at least one day although
N-acetylneuraminic acid gave only weak coloration with this reagent.
The advantage of this spray is that the plates do not have to be heated.
(2) Ammo&urn biwlfafe. Ziminiski (12) suggested the use of a non-
454 ANDREW E. GAL

FIG. 1. Reproduction of thin-layer separation of monosaccharides on silica gel G:

(A) galactosamine hydrochloride; (B) glucosamine hydrochloride ; (C) lactose ;
(D) galactose; (E) glucose; (F) N-acetylgalactosamine; (G) IV-acetylglucosamine.
Solvent system A: n-propanollwater (7/l v/v). 20 pg of the monosaccharides were
applied. This plate was visualized with the ammonium bisulfate spray. On this
particulate plate lactose was also spotted.
corrosive mixture of ammonium sulfate (20%) and sulfuric acid (4%).
We modified this reagent so that it would char all the monosaccharides
by simply using a 30% aqueous solution of ammonium bisulfate, and ob-
tained darker spots. The background stays white after heating for 20
min at 140°C.
(3) Aniline hydrogen phthalate. This is a 2.5% solution of aniline hy-
drogen phthalate in water saturated with n-butanol (13). It produces
less intense spots than silver nitrate. It is not suited for acetylated
sugars. Under UV light, 1 mg of neutral sugar could be detected after
heating for 15 min at 110°C.
(4) p-Aminob~ewoic acid. This reagent described by Roy (14) was
used with the following modification, Oxalic acid (2H,O), 75 mg, was
dissolved in 15 ml of ethyl alcohol. The resulting solution was added to
150 mg of p-aminobenzoic acid in 25 ml of chloroform and 2 ml of acetic
acid. When viewed in UV light, 0.5 ,pg of glucose or galactose could be
 THIN-LAYER CHROMATOGRAPHY OF MONOSACCHARIDES 455

Solvent Front

0
0 0
0

a
art gin
is c b E F G

FIG. 1. (Continued).

detected. It is not as intense with other sugars. Heating: 15 min at

110°C.
(5) Ninhydrin. The solution was prepared according to Sambasivarao
and McCluer (15) by dissolving 200 mg of ninhydrin in 95 ml of n-
butanol and 5 ml of pyridine. Heating the plates is not necessary. This
reagent will visualize only the amino sugars.
(6) EZson-Morgan reagent (10). Spray I: to 50 ml of n-butanol con-
taining 1% acetylacetone was added 40 mg of potassium hydroxide in
0.5 ml of 90% ethanol. Spray II: 1 gm of 4-dimethylaminobenzalde-
hyde. was dissolved in 60 ml of ethanol/hydrochloric acid (l/l) ; this
solution- was then diluted with 180 ml of n-butanol. The plates were
sprayed with Spray I and, after 5 min heating at 105”C, were sprayed
with Spray II and heated to 90°C. The N-acetylamino sugars give pur-
plish, the amino sugars pink, coloration. None of the other monosac-
charides could be detected with this reagent.
456 ANDREW E. GAL

FIG. 2. Reproduction of thin-layer separation of monosaccharides on silica gel G:

(A) galactose; (B) glucose ; (C) N-acetylgalactosamine; (D) N-acetylglucosamine.
Solvent system D: methyl acetate/isopropanol/water (l&/l/l v/v/v). 20 pg of the
monosaccharides were applied. This plate was visualized with the ammonium
bisulfate spray.
(7) Thiobarbituric acid spray reagent (7, 16). Spray I: sodium perio-
date, 0.5 M in 0.05 N sulfuric acid. Spray II: ethylene glycol, acetone,
and sulfuric acid (50/50/0.3 v/v/v). Spray III: sodium-a-thiobarbi-
turate, 6% in water. The plates were sprayed with Spray I and, after 15
min at room temperature, were thoroughly dried in a steam of air. The
plates were similarly treated with Spray II and this step was repeated
if the odor of formaldehyde persisted. After spraying the plates with
Spray III, they were heated at 100°C for 10 min. Only neuraminic acids
showed up as red spots.
Thin-Layer Chromatography of a Monosaccharide Mixture Originat-
ing from a Glycolipid. Human erythrocyte globoside, described by Yama-
kawa et al. (17)) 5 mg, was hydrolyzed with methanolic hydrochloric
acid and with hydrochloric acid according to Gallai-Hatchard and Gray
(2). An aliquot containing about 160 a of the liberated sugars was
spotted on a silica gel G plate. Glucose, galactose, galactosamine hy-
 THIN-LAYER CHEOMATOGRAPHY OF MONOSACCHARIDES 457

Solvent Front

;\ i E, ;,
origin

FIG. 2. (Continued).
drochloride, glucosamine hydrochloride, and N-acetylgalactosamine, 20
llg of each, were also spotted on the plate, which was developed with
n-propanol/water (7/l v/v).
RESULTS AND DISCUSSION
The RI value of the monosaccharides derived from glycolipids and
glycoproteins in the various solvent systems is shown in Table 1. These
values represent the average of a minimum of two determinations. The
same relative mobility of the various monosaccharides was obtained
when silica gel H or commercially prepared silica gel G plates (Anal-
tech) were used in the range from 0.5 to 30 fig of the individual sugars.
Saturation of the chromatography chamber with solvent vapors in general
lowers the Rf values of the sugars and was of no advantage.
Solvent system A is the most usefuI for survey of monosaccharides in
458 ANDREW E. GAL

FIG. 3. Reproduction of thin-layer separation of hydrolysis products of glycolipid

globoside: (A) galactose; (B) glucose; (C) mixture of monosaccharides derived
from hydrolysis of globoside; (D) galactosamine hydrochloride; (E) glucosamine
hydrochloride; (F) IV-acetylgalactosamine. Solvent system A: n-propanol/water
(7/l v/v). About 160 pg of the hydrolyzate and 20 gg of each monosaccharide
ztandard were applied. This plate was visualized with the ammonium bisulfate spray.

general (Fig. 1). Galactose is not separated from N-acetylglucosamine

in this system. Solvent system B allows the separation of galactose from
N-acetylglucosamine; however, in this system glucose and N-acetyl-
glucosamine have the same Rf value. Thus for complete identification
the chromatograms should be developed in both systems. Solvent system
C is well suited for the separation of amino sugars from the monosac-
charides. System D is particularly satisfactory for the. separation af
glucose and galactose (Fig 2) ; it takes less than 1 hr to, develop, but
it has to be dried and redeveloped:,.at least once. It is not satisfactory
for amino sugars. System E’ was developed for neuraminic acid separa-
tions. In this system the neutral sugars and the acylamino sugars move
with the solvent front. The spraying of the plates with the specific re-
agents indicated in Table 2’ is recommended.
Chromatographic separation of the monosaccharides originating from
human erythrocyte globoside is shown in Fig. 3. This glycolipid con-
 THIN-LAYER CHROMATOGRAPHY OB MONOSACCHARIDES 459

Solvent Front

FIG. 3. (Continued).

tains glucose, galactose, and N-acetylgalactosamine in a ratio of 1/2/l.

As the reproduction shows, N-acetylgalactosamine, deacetylated during
the hydrolysis, appears as galactosamine. The double galactose content
is clearly visible on the developed plate.

TABLE 1
R, Values of the Monosaccharides in Different Solvent Systems
Solvent systems
Compounds A B C D E

N-Acetylneuraminic acid 0.03 0.05 0.02 0.01 0.44"

Galactosamine-HCl 0.13 0.19 0.22 0.07 0.43
Glucosamine-HCl 0.21 0.26 0.31 0.10 0.54
Galactose 0.54 0.55 0.72 0.30 0.9-1.0
Glucose 0.62 0.61 0.75 0.42 0.9-1.0
N-Acetylgalactosamine 0.45 0.51 0.66 0.26 0.9-1.0
N-Acetylglucosamine 0.54 0.59 0.74 0.33 0.9-1.0
Development time 4 hr 4 hr 5hr 3 X 50 minb 4 hr

a N-Glycolylneuraminic acid, Rf = 0.41.

b After a single development the plates were dried and run again. Each run lasted
50 min.
460 ANDFEiW E. UAL

TABLE 2
Specificity of the Spray Reagents

Silver nitrate No heating + + + +

Ammonium Dark spots on white + + + +
biiulfate background
Aniline hydrogen Fluoresce + + + +
phthalate
p-Aminobenroic Fluoresce + + + +
acid
Ninhydrin Specificity +
Elson-Morgan Specificity + +
Thiobarbituric acid Specificity +

These systems readily separate nanogram quantities of isotopically

labeled materials. Evaluation of the plates could then be made by one-
or two-dimensional radioactive scanning or by other suitab,le techniques
(18).
SUMMARY
Thin-layer chromatographic methods are described for the separation
of the following monosaccharides : glucose, galactose, glucosamine, gal-
actosamine, N-acetylglucosamine, N-acetylgalactosamine and neuraminic
acids.
Neutral solvent systems and unaltered silica gel G plates were used.
The monosaccharides are separated sharply, which allows these methods
to be used for the determination of these compounds. The preparation
and use is discussed of seven facile spray reagents that afford further
specificity for detection and evaluation of the sugars at the microgram
level.
The feasibility of these procedures was demonstrated with a hydrol-
yzate of human erythrocyte globoside.
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 THIN-LAYER CHROMATOGRAPHY OF MONOSACCHARIDES 461

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