Thin-LayerChromatographyof Urinary Carbohydrates
A Comparative Evaluation of Procedures
D. S. Young and Agathe J. Jackson
Ten procedures for thin-layer chromatography
of carbohydrates
in urine
were compared. The procedures differ considerably in their sensitivity and
ability to separate sugars of clinical importance. A procedure was developed
that permitted several sugars to be identified in normal urine samples without prior desalting or concentration. Celite was used as adsorbent, anisaldehyde as location reagent. The procedure is simple and reproducible, and is
readily modifed
for quantification
Additional Keyphrases
TLC
spray
systems
sugars
of sugars by thin-layer
in normal
S by the excretion
disorders
are characterized
of abnormally
large quantities
of sugar in the urine. It has been recognized
only
recently
that normal urine contains
many sugars
at a low concentration,
the clinical significance
of
which has not yet been established
(1). TLC1 techniques are presently
available for detecting
sugars
in abnormal
samples,
but prior concentration
of
samples is required to detect the quantity
of sugars
normally
present in urine. To determine
the clinical significance
of small differences
in the usual
excretion
pattern
of carbohydrates
it is necessary
to develop a screening
technique
sensitive enough
to detect
small concentrations
of the common
urinary sugars.
The numerous
samples that a clinical laboratory
may be required
to examine
necessitate
use of
unidimensional
chromatography
without
prior
desalting
or concentration
of samples.
In this
EVEItAL
METABOLIC
From
the Clinical
Pathology
i)epartment,
Clinical
Center,
National Institutes of Health, Bethesda, Md. 20014.
This paper was reviewed before submission.
Received
and accepted
Sept. 10, 1970.
1 Abbreviation
used:
TLC,
thin-layer
chromatography
(-graphic).
954 CLINICAL
and pancreatic
compared
#{149} semiquant
ifi cation #{149} Celite
results by column chromatography compared
CHEMISTRY,
Vol. 16, No. 11, 1970
densitometry.
disease
adsorbent,
#{149} 10
anisaldehyde
urine
study,
we compare
the separation
of carbohydrates
by several different
TLC procedures
(not
necessarily
originally
proposed
for physiological
fluids) with a technique
that we have modified
from that of Garbutt
(2), which we found to be
most suitable for identifying
and estimating
carbohydrates
in urine. The different
systems
were
evaluated
for their ability to adequately
separate
urinary sugars in a single dimension with minimum
sample
preparation.
The ease with which they
could be measured
by thin-layer
densitometry
was
also evaluated,
as was the reproducibility
of the
separation
and interference
from noncarbohydrate
constituents
of urine.
Materials and Methods
Reference System
Glass plates. Plates (20
with a Desaga
apparatus
P.O.
Box 407, Heidelberg,
adsorbent
was Filter-Cd
Co., Celite Division,
New
a thickness
of 250 u. For
plates, 15 g of Celite was
X 20 cm) were prepared
(C. Desaga
GmbH,
West Germany).
The
(Celite, Johns-Mansville
York, N.Y.), spread to
the preparation
of five
ground with 2 g of an-
Table 1. Characteristics of Evaluated TLC Systems
System
Reference
Adsorbent
Solvent
(proportions
by volume)
Location
reagent
A
Reference System
Celite
Ethyl acetate: pyridine:water
(60:25:20)
Anisaldehyde/H,S04
B
Garbutt(2)
Celite
Ammoniacal
C
Eastman
Kodak
n-Butanol: pyridine:water
(75: 15:10)
Ethyl acetate: methanol:acetic
D
Eastman
Kodak
E
Anderson and
Stoddart (4)
F
Hay
Silica-gel Chromagram
with sodium bisulfite
acid:water (60:15:15:10)
Acetone: chloroform: methanol:
Silica-gel
Chromagram
with sodium acetate
water (80:10:10:5)
Silica-gel Chromagram
#{128}1
at. (s)
G
Lato et at.
H
Adachi (7)
Silica gel
Silica gel
(6)
Silica gel
Paget and
J
Coustenoble
Wolf rom et al.
Silica gel
(8)
(9)
Cellulose
MN 300
hydrous CaSO4 in a mortar. Aqueous sodium acetate, 60 ml of 20 mmol/liter
solution., was added
and mixed for 1 mm in a Waring Blendor.
The
plates (now commercially
available from Analtech
Inc., Wilmington,
Del.) were poured immediately,
dried with a hot-air fan, and stored under reduced
pressure
in a dessicator.
They were activated
before use by heating at 100#{176}C
for 30 mm.
Marker
solution.
D-Glucuronic
acid, lactose,
maltose,
sucrose,
D-galactose,
D-glucose,
D-fructose, D-mannose,
L-arabinose,
D-ribose,
and Dxylose, 100 mg of each, were dissolved in 100 ml of
water. The mixture
was stored in small aliquots
at -20#{176}C.
Quantity of urine to be chromato graphed. Because
the concentration
of salts has a considerable
influence on Rf values of sugars, the volume of urine
applied to the plate was determined
by the specific
conductivity
of the sample,
as measured
on a
conductivity
bridge,
Model 31 (Yellow
Springs
Instrument
Co., Yellow Springs, Ohio). Different
volumes were applied to the plate according to the
schedule:
specific conductance
greater
than
22
mmhos/cm,
2.5
of urine was applied to the plate;
between
15 and 22 mmhos/cm,
5.0 1d; and between 10 and 15 mmhos/cm,
12 zl. Samples were
applied as streaks
1-cm long and 1-cm apart. A
microtitrator
(Beckman
Instruments
Inc., Fullerton, Calif.) was used to apply the sample.
Solvent.
A mixture
of ethyl acetate,
pyridine,
and water (60:25:20,
by volume)
was prepared
freshly for each migration.
The time required
for
10-cm migration
in “sandwich”
chambers
(Brinkmann
Instruments,
Inc., Westbury,
N.Y.)
was
about 75 mm. After the solvent had migrated
10
cm, the plates were dried with a cool-air fan until
Butanone :acetic acid: water
(3:1:1)
1-Butanol : acetic acid: ethyl
ether:water (9:6:3:1)
n.Butanol:ethyl
acetate: isopropanol:acetic acid:water
(35:100:65:35:30)
n-Propanol:water
(17:3)
n-Butanol: methanol: water
(5:3:1)
Ethyl acetate: pyridine:water
(2:1:2) (upper phase)
AgNO,
Aniline hydrogen
phthalate/CH,COOH
Aniline hydrogen
phthalate/CH,COOH
Naphthoresorcinol/H,S04
Resorcinol
in phosphoric
acid/H2S04
Naphthoresorcinol/H,504
Thymol/H,S04
Thymol/H,504
Aniline hydrogen
phthalate/CH,COOH
the smell of pyridine
had disappeared.
Although
separation
by a single migration
was adequate,
a
second migration
with fresh solvent improved
the
resolution
of the sugars.
Location reagents. For detection
of the sugars,
the dried plates were sprayed with about 5 ml of
a mixture
of 9 ml of absolute
ethanol,
0.5 ml of
concentrated
sulfuric acid, and 0.5 ml of anisaldehyde. The plates were then heated at 100#{176}C
until
the color of the spots was maximum
(20 to 30 mm).
For measurement
of the sugars, p-aminobenzoic acid was prepared
as described
by Saini (3)
and used as a spray. Reddish-brown
spots were
produced
with all carbohydrates
when the plates
were heated to 105#{176}
to 110#{176}C.
The sensitivity
of
this reagent was comparable
to that of anisaldehyde. The sugars were estimated
by scanning the
plates in a Spectrodensitometer
(Farrand
Optical,
Mt.
Vernon, N.Y.)
at 550 nm.
If the excretion
of a sugar appeared
to be abnormally
high, the stock sugar was diluted with
water, to contain 0.5 and 5.0 /.Lg/J.Ll. These were
applied as streaks of 10
The absorbance
of the
sugar in the urine sample could be compared
with
that of the standard
solutions to gain a semiquantitative estimate of its concentration.
Evaluation
Procedure
Nine different systems as described in the literature were compared
with the Reference
System
(Table 1). The procedures
were followed as recommended except that Brinkmann
sandwich
chambers were used throughout
for all plates
and
Chromagram
sheets
(Eastman
Kodak
Co.,
Rochester,
N.Y.). The same system was run in
duplicate
on three different
days and the same
CLINICAL
CHEMISTRY,
Vol. 16, No. 11, 1970 955
Results
samples were run on each occasion. The samples
were:
#{149}
3 zl of the 11-sugar “marker”
solution listed
above.
#{149}
3,.d of five “clinically
important”
sugars
(glucose, lactose, galactose,
sucrose, and fructose)
in water.
#{149}
Urine I, from a normal subject (with specific
conductance
25.7 mmhos/cm).
#{149}
Urine II, from a patient
with pancreatic
disease, containing
several sugars in abnormal
concentration
(specific
conductance,
20.6 mmhos/
cm).
The two urine samples
were applied
without
desalting.
Individual
sugars in water were run to
confirm the identity
of sugars in the mixtures
if
the sugars did not yield characteristic
colors, or if
separation
was inadequate.
Three additional
systems
(10, 11, 12) were investigated
but found to be unsatisfactory
(see
below).
All systems were used according
to the
published
description.
If the location reagent was
unavailable
or unsatisfactory,
other location
reagents were evaluated.
The ambient
temperature
during all experiments
varied between 24.5#{176}
and
27#{176}C.
System
J (Table 1) was run both in sandwich
chambers
and a tank. Only the Rf values from
runs in a tank are reported
because the solvent
separated
into two phases in the sandwich
chambers, and the spots were less well resolved.
Main differences
between
the operating
conditions of the 10 systems are summarized
in Table 2.
Because
of the different sensitivities
of the location
reagents,
volumes of marker solutions
and urine
were adjusted to ensure reasonable
clarity of spots.
Rf values of the sugars in the 11-sugar marker
solution
are listed in Table 3, and represent
the
mean value for duplicate
runs on three different
days. The color of the sugar spots with the various
location reagents is also included.
Separation
capabilities
of the different
systems
are summarized
in Table 4. The location reagents,
or their mode of application,
had to be modified
from the original description
for several systems as
indicated
below.
Comparison of Systems
System A. The 11 sugars in water separated
into
nine clear bands (only the resolution
of ribose from
xylose, and of fructose
from mannose
being inadequate).
Rf values of the sugars in undesalted
urine were about 5% less than in water. However,
the distinctive
colors with anisaldehyde
helped us
identify
the sugars without
difficulty.
Urea gave
a yellow spot, fading to white, with an Rf value
slightly
greater
than that of ribose. Uniformly
thick plates were hard to prepare.
System B. Only five bands could be identified
Table 2. Operating Conditions of Different TLC Systems for Sugars, as Required for Optimum
Separation and Sensitivity of Sugars
System’
Migration
distance,
A
cm
10
Development time, mm
75 x 2
Thickness of adsorbent, z
Volume marker solution, l
Volume undesalted urine, Ll
250
3.0
2.5
B
C
D
E
F
G
H
12
280
250
3.0
2.5
10
70
100
3.0
2.5
10
35
100
3.0
5.0
12
80
100
10.0
10.0
12
165
200
3.0
5.0
12
215
300
5.0
10.0
13
295
250
5.0
5.0
J
12
100
300
3.0
5.0
12
25 x 2
500
10.0
10.0
See Table 1.
Table 3. Migration Distances and Reaction with Location Reagent Compared for Systems in Table 1”
System
Carbohydrate
Ribose
Xylose
Arabinose
Mannose
A
B
71 GyG
70 fYG
58Y
53YG
30 YBr!
29 YBr!
58V
63V
40 RBr!
21 YBr!
34Y!
16YBr!
32 BrY!
14 Br!
llBr!
52B
55V
62 V
52 V
45 fV
36 V
22 V
8 Pk
0V
100
54 B
52 B
47 B
468
43 R
41 B
39 B
34 R
52 RBr!
46 RBr!
46 B
39 G
31 B
27 B
19 GB
valuesare Rf values X
E
51 V
Fructose
tion reagent; d, dark; f, faint;
D
73V
Glucose
Galactose
Sucrose
Maltose
Lactose
Glucuronic acid
C
31 BrY!
13 Y!
19 Y!
4 Y!
0 BrY!
28 B
43 RP
56 RP
43 RP
44 RP
36 RP
41 RP
34 RP
30 RP
24 RP
16 RP
11 RP
G
H
64 B
59 GyP
63 B
65fP
54 B
55 B
52 R
52 GyV
45 GyV
41 R
35 Pk
54 P
26V
36 BV
58Br
56 P
55 P
49 P
52 P
47 P
34 P
21 fBr
and represent the mean of duplicates on three different days. Color reactions
I
25GyB
48 GyB
52 GyB
50 Br
J
63R
53R
47 R
46dG
20 Pk
50 Pk
42 Pk
4idG
36dG
61 Pk
58 Pk
41 Pk
9 Gy
29fPk
23dG
12Or
not visible with loca-
!, fluorescent; Pk, pink; G, green; B, blue; Y, yellow; Gy, grey; Br, brown; R, red; P. purple; v, violet; Or.
orange.
956
25B
228
F
CLINICAL CHEMISTRY, Vol. 16, No. 11, 1970
System”
ABCDEFGH
I
J
Number of discrete bands
from 11-sugar mixture
9 5 0b 0 5 4 5 7 4 8
Number of discrete bands
from 5-sugar mixture
5 3 2 2 4 3 3 3 3 3
Additional
bands in urine I
not due to reference
sugars
1 1 2 2 1 1 2 1 1 1
“See Table 1.
A listing of 0 indicates that a continuous streak was present
without clear differentiation
of separate components.
the 11 sugars in water. Only glue uronic acid,
lactose, maltose, and xylose separated
as discrcte
bands;
the other sugars remained
in a continuous
streak.
The five-sugar
mixture
separated
into
three bands. Sucrose stained very weakly with the
recommended
location
reagent
(silver nitrate
in
ammonia).
Migration
in sandwich
chambers
required 4.5 h, in contrast to the 0.5 h cited by the
author, who used a tank.
System C. Maltose,
sucrose, and fructose failed
to yield a visible or fluorescent
spot on spraying
with the location reagent (aniline hydrogen phthalate in glacial acetic acid). Urine samples produced
one yellow fluorescent
band and one visible pink
band, which could not be correlated
with any
known sugars.
System
D. The same criticisms
apply to this
system as to System C. The low Rf values of all
sugars further
impaired
the resolution
of bands
and rendered
tentative
identification
by Rf more
difficult.
System E. The separation
of the 11 sugars in
water was poor. The recommended
location
reagent (ethanolic
aniline oxalate)
yielded
yellow
smudges
and protracted
heating
of the sheets
melted them. The use of napthoresorcinol
in ethanol and concentrated
sulphuric
acid enabled five
bands to be identified
from the 11-sugar marker
solution,
and four bands from the five-sugar
solution. Separation
of the carbohydrates
in urine was
poor. Pink and grey bands were produced
by the
urine samples, which could not be correlated
with
known sugars.
System
F. Only four bands were identifiable
when the 11-carbohydrate
solution
was applied,
and three were identified in the five-sugar solution.
Urine specimens yielded three bands, one of which
had a greater Rf value than any of the sugars in
the marker solutions.
One of the other spots was
due to xylose, and the third was very diffuse and
could have contained
glucose, galactose,
fructose,
mannose,
arabinose,
or ribose. Sulfuric acid, used
by us as location reagent, gave faint brown spots
on heating
but, when resorcinol
(20 g/liter)
in
from
acid
was next
applied,
strong
redpurple
spots appeared
on a yellow-pink
background. Glucose and fructose were better separated
in the sandwich
chambers
than described
in the
original
description,
in which a tank was used.
System G. Five bands were separated
from the 11carbohydrate
solution,
three from the five-component solution. Glucose and fructose had identical Rf values. With urine, additional
spots appeared above the sugars of greatest
Rf value, but
their identities
were not detemined.
The adsorbent-silica
gel without
binder-tended
to flake
and become powdery,
producing
uneven solvent
fronts.
System
H. Seven bands were separated
from the
11-sugar
marker
solutions,
three from the fivesugar solution. Urine samples produced two spots;
one was glucose,
the other migrated
further than
any marker
sugar.
Glucose
and fructose had similar Rf values. The strength of the location reagent
had to be increased
over the recommended
concentration
to produce strong spots.
System I. Because of streaking on the plate, only
three distinct
bands were discernible
from the
marker solutions.
The location reagent gave weak
colors with all the sugars and galactose
was poorly
separated
from lactose.
System J. Eight spots were produced
from the
11-sugar
marker
solutions,
three from the fivesugar solution when a tank was used. As with systems C and D, aniline hydrogen
phthalate
in glacial acetic acid failed to make fructose and sucrose
visible at the concentrations
present.
Sugars in
urine were incompletely
separated
but desalting
improved the resolution.
Cellulose MN 300 (Brinkmann) was used in preference
to Avicel (recommended by the authors) because of difficulty in prephosphoric
Table 4. Apparent Separation Capabilities
of Different Systems
paring
uniformly
Other systems.
coated
plates.
Even
when 15 il of urine and
marker
solutions
was applied
to the plate in the
system
described
by Becker and May (10), the
spots that developed
were very faint, and the carbohydrates
in the mixtures
failed to separate.
The system described
by Kudla and McVean (11)
was rejected
because
of the need for continuous
migration
to obtain adequate
separation
of sugars,
as the solvent front travels
much more rapidly
than any of the sugars. The system did produce
good separation
of the sugars in both marker solution and urine, although
the colors produced
by
reaction
of the location
reagent
with the sugars
faded within 5 mm. Permanent
colors and a welldefined solvent front are necessary
requirements
of a system
for identification
and measurement
of
sugars.
The location reagent used in the original method
of Vomhof
and Tucker (12) is no longer commercially available.
Other location reagents
failed to
demonstrate
clearly the sugars in the reference
mixture,
although
the same reagents
had proved
CLINICAL
CHEMISTRY,
Vol. 16, No. 11, 1970 957
Table 5. Carbohydrates in Two Undesalted Urines, as Provisionally Identified
by Use of Different Systems”
System
A
B
D
E
F
G
H
I
.1
Urine I
D,F,GA
D,GA,L,X
GA
GA
0
Urine II
D,F,GA,S
D,F,GA,L,X
GA
?D,GA
D
X
X
D,GA
D,G,GA
D,X
D,X
0
D,F
GA
D,X
D, glucose;F, fructose;
successful
with
G, galactose;
the other
C
GA, gl ucuronic acid; L, lactose; 5, sucrose; X, xylose.
systems
discussed
pre-
viously.
Compounds Detected in Urine
A tentative
identification
of the carbohydrates
detected
in undesalted
urine from normal subject
I and from patient II (with pancreatic
disease), as
indicated
by the different
systems,
is listed in
Table 5. There was little difference
between
the
compounds
identified
before and after desalting,
although
galactose
and lactose were detected
in
urine II, when system
A was used after desalting.
When urines I and II were high-pressure
colunm
chromatographed
according
to the procedure
described by Jolley and Freeman
(1), many additional carbohydrates
were detected
in both samples. The column
chromatographic
system
was
used to determine
the concentration
of the sugars
detected
by the thin-layer
chromatographic
systems (Table 6). Glucuronic
acid is not included, as
it has not yet been possible to quantify
it with the
column-chromatographic
system.
Discussion
Requirements
for a thin-layer
chromatographic
system
for urinary
sugars differ, depending
on
whether the objective
is to detect one compound
that is present
in great
excess
or many
sugars
present in their normal concentrations.
When the
latter is important
essential
considerations
are the
adequate
separation
of the compounds
and sensitive location reagents.
The sugars we included
in
the five-component
marker solution are important
in the metabolism
of carbohydrates
in humans.
Table 6. Approximate Concentrations of Sugars
(mg/liter) in Urines I and II, as Determined
by High-Pressure Column Chromatography
Urine
Sucrose
40
Lactose
Fructose
Galactose
Xylose
10
10
20
25
Glucose
150
I
Urine
II
Normal
range
200
80
0-150
0-100
170
40
90
0-50
0
570
(IS)
0-30
10-120
958 CLINICAL CHEMISTRY, Vol. 16, No. 11, 1970
should isolate these
sugars from each other and from ribose and arabinose, which are also normal urine constituents.
Even with compact spots a difference of 0.03 between Rf values of two sugars is necessary
for
clear differentiation,
so that tentative
identification may be assigned. System A produces adequate
separation
of the important
carbohydrates,
although ribose is not well separated
from xylose,
nor fructose from mannose.
However,
the difference in color produced
by reaction of these compounds with anisaldehyde
helps in identification.
The other systems
either failed to separate
the
sugars adequately
(as with Systems
B,E,F,G,H,
and I), or the location reagent failed to react with
some of the important
sugars (Systems
C,D, and
J).
Location
reagents
such as anisaldehyde,
which
yield different
colors with different
sugars,
are
useful because
both characteristic
color and Rf
value are useful in provisional
identification
of the
compounds.
For quantification
by densitometry
a
uniformly
colored spot against a light background
is desirable.
Standards
of all the sugars to be measured should be included in the same run. Mes and
Kamm (14) have recently
compared
the sensitivity of various location reagents for carbohydrates.
Their study indicated
a large difference
between
sensitivities,
which partly accounts
for the different sugars found in the urine samples in this study
(Table 5). The combination
of adsorbent
and location reagents used for Systems A and B allowed a
greater
number
of sugars to be identified
than
with any other system except J, for which four
times the volume of sample was used.
Because different chromatographic
systems and
location
reagents
do not necessarily
reveal the
same carbohydrates
in the same sample,
caution
must be used in interpreting
results.
Additional
separations
with different location reagents should
be performed
before definitive identities
should be
ascribed
to provisionally
identified
compounds.
This study has demonstrated
that it is possible to
separate
several
different
carbohydrates
from a
small volume of normal
urine. This permits
the
Adequate
development
separation
systems
of a systematic
approach
for
rapid
screening of urine for small variations
in composition of carbohydrates
so that abnormal
samples
can be identified
for study in greater
depth by
more sophisticated
procedures.
References
8. Paget,
M., and Coustenoble,
P., La chromatographie
sur
couches
minces des oses et osides rencontr#{233}sdans des enzymopathies affectant.les glucides.Ann. Biol. Clin. 25, 1239 (1967).
1. Jolley, R. L., and Freeman,
M. L., Automated
carbohydrate
analysis of physiologic fluids.CLIN. CHEM. 14, 538 (1968).
2. Garbutt, J. L., Inexpensive
adsorbents for thin-layer chromatography
of carbohydrates.
J. Chromatogr.
15, 90 (1964).
3.
Saini, A. S., Some technical
improvements
in the paper chromatography
of sugars. A method
of sample desalting
and a sensitive staining
reagent.
J. Chromatogr. 24, 484 (1966).
4. Anderson,
D. M. W.,
sugars on “Chromagrams.”
5. Hay,
tography
(1963).
G. W., Lewis,
in the study
and Stoddart,
Carbohydrate
B. A., and Smith,
of carbohydrates.
J. F., Separations
Res. 1, 417 (1966).
F., Thin-film
of
chroma-
J. Chronw.Iogr. 11, 479
6. Lato, M., Brunelli,
B., Ciuffini, G., and Mezzetti,
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