Planar Chromatography in Practice

The fingerprint of biopolymers

(polysaccharides)
Standard solutions
The sugars or uronic acids were weighted (10 mg
each) in 10 mL reaction vials each and dissolved in
1 mL methanolic hydrochloric acid (0.5 or 2 mol/L
[1]). After methanolysis at 100 °C for 4 h, 50 µL
pyridine were added. Then, two mixtures were com-
posed in 2 mL measuring flasks by adding 30 µL
each (Fructose 90 µL) and filling up to the mark
(150 ng/µL; fructose 450 ng/µL).

20
F. Gamlich and D. Schick

An HPTLC method for rapid analysis of plants’

polymers was developed at the Institute of Food
Chemistry, University of Hohenheim, Stuttgart,
Germany, in cooperation with food technologist
Frank Gamlich during an internship supervised by
Prof. Morlock, and Dinah Schick during her diploma
thesis supervised by Prof. Schwack.

Introduction
Most biopolymers in plants are polysaccharides,
which have distinct properties: They bind water
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and have energy storage or structural capacities.
Polysaccharides are isolated from terrestrial plants, After methanolysis, both mixtures mainly showed
and seaweed, but also organic derivatives are on the the methyl glycosides and methyl glycoside methyl
market. In nutraceuticals, food, pharmaceuticals or esters formed from sugars and uronic acids, respec-
other products, they are used as thickening agents tively. But also unknown side products were formed
to increase the viscosity, as hydrocolloids to build (marked*).
stable gels or as stabilizers to improve suspensions
or emulsions. Sample preparation
The analysis of thickening agents, generally The thickening agents and food samples were pre-
performed by GC [1], is challenging. An HPTLC pared according to the protocol [1].
method complementary to GC was developed
and validated with which the different types Sample application
of thickening agents can readily be differenti- Bandwise with Automatic TLC Sampler 4, 21 tracks,
ated. The accuracy of the results obtained by band length 8 mm, track distance 9 mm, distance
this highly effective, rapid and cost-saving new from lower edge 8 mm, application volume 1–7 µL
HPTLC method was successfully verified by for samples and 2–15 µL for standards.
comparison with the GC method.
Chromatography
Chromatogram layer In the ADC 2, with 10 mL i-propyl acetate – ethyl
HPTLC plates silica gel 60 (Merck), 20 x 10 cm acetate – methanol – water 5:4:1:0.1 (v/v/v/v);

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migration distance 60 mm; drying time 30 s before after derivatization with the aniline diphenylamine
and 2 min after development o-phosphoric acid reagent. Alginic acid and its
sodium, potassium and ammonium salts (tracks a
Densitometry to d, respectively) showed the same pattern, which
TLC Scanner 3 with winCATS software, spectra record- slightly differed due to natural variances. Only pro-
ing from 200–800 nm, absorption measurement at pylene glycol alginate showed a different pattern
370 and 630 nm (multi-wavelength scan), slit di- because of its typical organic rest (track e). Agar
mension 6 mm x 0.45 mm, scanning speed 20 mm/s, agar and carrageen consist of the same monomeric
peak area evaluation by linear or polynomial units, so their patterns were almost the same (tracks
regression f and g, respectively). But the galactomannans, i.e.
carubin and guaran, clearly differed in the ratio of
Post-chromatographic derivatization mannose to galactose (~ 4:1 for carubin and ~ 2:1
The HPTLC plate was immersed in the aniline diphe- for guaran, tracks h and I, respectively) and could
nylamine o-phosphoric acid reagent (20 % o-phos- be differentiated, although they contain the same
phoric acid (85 %) were added to the 1:1 mixture monomeric units. A mixed powder of guaran and
of 2 % solutions of diphenylamine and aniline in microcrystalline cellulose is also depicted (track j).
acetone) using the TLC Immersion Device (immer- Plant exudates (traganth, gummi arabicum, karaya;
sion time 1 s, speed 3.5 cm/s) and heated on the tracks k, l and n, respectively) could clearly be
TLC Plate Heater at 110 °C for 5 min. The reagent distinguished. Track m illustrates the bacterial-de-
stored in the refrigerator was stable for months. rived xanthan gum for comparison. The pattern of
various pectins was characteristic (track o: pectin c,
Documentation track p: pectin a, track q: pectin not specified) and
The chromatograms were documented under white could even be differentiated to formulations with a
light illumination (transmission mode) using the low pectin content (only 20 % in the formulation,
TLC Visualizer. track p*), which showed arabinose as the main
monomeric unit. Glucose is the monomeric unit of
Results and discussion microcrystalline cellulose and starch, which showed
After a reduced sample preparation, the separa- the same patterns (tracks t and u, respectively).
tion of 21 samples, i.e. the methyl glycosides and Sodium carboxymethyl cellulose (Na-CMC, track r)
methyl glycoside methyl esters of the different showed an additional band above, whereas hy-
plant polysaccharides as well as standard mixtures, droxypropyl methylcellulose (HPMC, track s) was
took only 20 min. The plates were documented characterized by a complex pattern. To conclude, all

HPTLC separation of different plant polysaccharides after methanolysis showing their monomeric units (discussed above); Mix 1 (x) and Mix 2 (y)

10 CBS 108
plant polysaccharides based on different monomeric way as GC for quantification. The sample number
units showed characteristic fingerprints and could varied depending on the polysaccharides available
be differentiated by the new HPTLC method. by donation from C.E. Roeper, Biesterfeld, FMC
For food analysis, exemplarily shown for milk and BioPolymer, Provisco, AppliChem and Harke.
apple juice, the plants’ polysaccharides were iso-
Analysis by GC HPTLC
lated and methanolyzed. Marker compounds were Polysaccharide Monomeric units Mean recovery Mean recovery
n n
selected (red) for quantitation and the absorbance rate ± SD (%) rate ± SD (%)
Carubin Gal, Man, (Ara) 4 82 ± 12 8 108 ± 26
measurement was performed at 370  and 630 nm Guaran Gal, Man, (Ara) 7 90 ± 36 12 87 ± 36
after spectra recording. In case marker substances Traganth GalA, Xyl, Fuc, Gal, Ara 3 51 ± 12 4 44 ± 3
Gummi arabicum GlcA, Man, Gal 4 78 ± 16 4 99 ± 13
were not available (3,6-anhydrogalactose, man- Xanthan GlcA, Man, Glc 4 65 ± 13 6 54 ± 13
nuronic (ManA) and guluronic acid (GulA)), the Pectin GalA, Gal, Ara 16 53 ± 22 23 83 ± 20
most intensive spot was used for quantitation. Alginic acid ManA, GulA 4 38 ± 22 4 35 ± 12
Alginates ManA, GulA 14 66 ± 30 16 80 ± 33
Agar agar Gal, 3,6-AnhydroGal 4 54 ± 19 4 41 ± 7
agar agar gummi arabicum Carrageen Gal, 3,6-AnhydroGal 8 117 ± 33 8 86 ± 6
calibration samples calibration samples

To conclude, the results obtained by HPTLC and GC

are comparable. However, the HPTLC method is
more effective in terms of sample throughput, ro-
bustness, costs and especially analysis time. With
the same number of samples, the HPTLC method is
about 8 times faster than the GC method.

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[1] BgVV, Amtliche Sammlung von Untersuchungsverfahren
Analysis of agar agar isolated from apple juice (left; calibration nach § 64 LFGB, Methode L 00.00-13, Beuth Verlag, Berlin,
below) and of gummi arabicum isolated from milk (right) Köln, November 1986.
Further information is available from the author on
For verification of the results, HPTLC was compared
request.
with GC, for which an additional silylation was re-
Contact: Prof. Dr. Gertrud Morlock, Justus-Liebig-University
quired [1]. The mean recovery rates of plants’ poly- of Giessen, Institute of Nutritional Science, IFZ, Heinrich-Buff-
saccharides isolated from milk (blue) and apple juice Ring 26, 35392 Giessen, Germany, Gertrud.Morlock@ernaehrung.
(orange) showed that HPTLC is suited in the same uni-giessen.de

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