A-07.3 Determination of Mono-, Di-, Tri-And Polysaccharides

APPLICATION NOTES »INSTRUMENTAL THIN-LAYER CHROMATOGRAPHY«
Determination of Mono-, Di-, Tri- and Polysaccharides A-07.3
Key words
Instrumental HPTLC - quantitative analysis - qualitative identification - densitometry (absorbance) -

post chromatographic derivatization - food analysis - process control - carbohydrates
Scope
The analysis of sugars, e.g. in various food stuff, is important for manufacturer, authorities and
private investigation laboratories. This analysis can easily and rapidly be performed by planar chromato-
graphy.
Depending on the mixture of sugars in a sample 3 different separation systems can be employed.
The extracts are chromatographed on silica gel or amino plates, respectively without prior derivati-
zation. Post chromatographic derivatization with diphenyl amine-aniline-phosphoric acid-reagent
follows for all standards and samples simultaneously. Using the amino plate the sugars fluoresce by
simply heating the plate. Densitometric evaluation is done by absorbance measurement at 620 nm
or by fluorescence measurement at 366/>400 nm. The limit of detection for fluorescent zones is
about 10 ng.
Literature
R. Gauch, E. Leuenberger, E. Baumgartner, J. Chromatogr. 174, 195-200 (1979)

R. Klaus, W. Fischer, H.E. Hauck, Chromatographia 28, 364-366 (1989).
K. Patsch, S. Netz, W. Funk, J. Planar Chromatography 1, 39-45 (1988), Part 1.
K. Patsch, S. Netz, W. Funk, J. Planar Chromatography 1, 177-179 (1988), Part 2.
Advantages of using HPTLC for this analytical task
• Simple sample preparation, no pre-chromatographic derivatization

• Simultaneous quantitative determination of several saccharides in contrast to
enzymatic methods
• High sample throughput
6/97 A-07.3 1 (4)

Chemicals
Acetone
Acetonitrile
Water dist.
Diphenylamine
Aniline chloride Standards:
Methanol Raffinose Sucrose
Butanol Melezitose Galactose
Phosphoric acid, 86 % Lactose Glucose
Boric acid, 0.5 % Maltose Fructose
Sample preparation
Depending on type of foodstuff dissolve or extract sample with water, and filter if necessary. Co-
extracted proteins, which would interfere with chromatographic analysis, can be precipitated by
adding twice the amount of acetone (cold).
Dilute the solutions with methanol to a final concentration of about 0.2 mg/mL per saccharide.
Standard solutions
Dissolve 0.5 mg of those saccharides which shall be determined in 10 mL dist. water and dilute 1:4
with water (125 ng/µL).
Chromatogram layers
1) To separate mono-, di- and trisaccharides: HPTLC Silica gel Merck 60 F , 20x10 cm
254
2) To separate polysaccharides: HPTLC Silica gel Merck 60 (without F), 20x10 cm

3) To separate glucose from fructose: HPTLC Merck NH2 F 254s, 20x10 cm
Sample application
With CAMAG Linomat as 7 mm bands, track distance 3 mm, distance from left edge 20 mm, dis-
tance from lower edge 8 mm; delivery rate 15 s/µL = 17 applications per plate.
Application scheme:
S1 U1 U2 S2 U3 U4 S3 U5 U6 S4 ...
2 2 2 3 2 2 4 2 2 5 ... µL/band
S1- S4 standards in different concentrations; U unknowns
Alternatively the Automatic TLC Sampler can be used.
6/97 A-07.3 2 (4)

Chromatography
In CAMAG Twin Trough chamber 20x10 cm

1) To separate mono-, di- and trisaccharides, except glucose from fructose:
- 10 min conditioning with developing solvent.
- Develop three times with acetonitrile - water 85:15; migration distance 70 mm each.
- Dry with hair drier held at a distance of about 40 cm for 20 min (at 40-45°C). After final run,
dry in the same way or at 100°C for 2 min in an oven.
2) To separate polysaccharides:
- 10 min conditioning with developing solvent.
- Develop with butanol - methanol - water 50:25:20; migration distance 70 mm.
- Dry plate for 15 min with hair drier.
3) To separate glucose from fructose:
- Develop with acetonitrile -water - 0.5% aqueous solution of boric acid 76:24:10; migration
distance 75 mm.
- Dry plate for 5 min with hair drier.
Derivatization
- By dipping for 3 s with CAMAG Chromatogram Immersion Device in diphenylamine reagent (2.4
g diphenylamine + 2.4 g aniline chloride in 200 mL methanol, then add 20 mL 86% phosphoric
acid*) followed by heating at about 120°C for 10-15 min.
- Using NH2-plates the sugars fluoresce simply by heating the plate at about 130°C for 5 min.
(Some sugars may require a higher temperature and time.)
Note: Dipping the plate into a solution of paraffin/n-hexane 1:2 enhances the fluorescence intensity
by a factor 2-3.
Densitometric evaluation
With CAMAG TLC Scanner and CATS evaluation software; scanning by absorbance at 620 nm with
tungsten lamp or by fluorescence at 366/>400 nm with mercury lamp, slit dimension 0.2x4 mm,
detection limit for fluorescent zones about 10 ng/zone.
* For the NH2-plate use acetone as solvent; the solution is stable in the dark for several weeks.
6/97 A-07.3 3 (4)

2 6
1 3
4 5 1 raffinose
2 lactose
3 maltose
4 sucrose
5 galactose
6 fructose and glucose
Fig. 1: Densitogram by 620 nm of 7 different saccharides (plate/chromatography see 1.)
6
5 1 - 5 polysaccharides
1 2 4
6 maltose
7 glucose
3
Fig. 2: Densitogram by 620 nm of an unknown sample of polysaccharides; maltose and

glucose (plate/chromatography see 2.)
3
2
1 1 raffinose
5 2 lactose
3 sucrose
4
4 glucose
5 fructose
Fig. 3: Densitogram by 620 nm of 5 different saccharides (plate/chromatography see 3.)
6/97 A-07.3 4 (4)

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APPLICATION NOTES »INSTRUMENTAL THIN-LAYER CHROMATOGRAPHY«

Determination of Mono-, Di-, Tri- and Polysaccharides A-07.3

Instrumental HPTLC - quantitative analysis - qualitative identification - densitometry (absorbance) -

R. Gauch, E. Leuenberger, E. Baumgartner, J. Chromatogr. 174, 195-200 (1979)

Advantages of using HPTLC for this analytical task

• Simple sample preparation, no pre-chromatographic derivatization

6/97 A-07.3 1 (4)

2) To separate polysaccharides: HPTLC Silica gel Merck 60 (without F), 20x10 cm

Alternatively the Automatic TLC Sampler can be used.

6/97 A-07.3 2 (4)

In CAMAG Twin Trough chamber 20x10 cm

6/97 A-07.3 3 (4)

Fig. 1: Densitogram by 620 nm of 7 different saccharides (plate/chromatography see 1.)

Fig. 2: Densitogram by 620 nm of an unknown sample of polysaccharides; maltose and

Fig. 3: Densitogram by 620 nm of 5 different saccharides (plate/chromatography see 3.)

6/97 A-07.3 4 (4)

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