TITLE: The identification of the sugars in fruit juices using TLC (thin layer

chromatography)

AIM: To identify the sugars in fruit juices using thin layer chromatography

INTRODUCTION

In this experiment we are identifying the sugars in fruit juices using

thin layer chromatography. Firstly, what is meant by thin layer chromatography?
Thin layer chromatography (TLC) is a chromatographic technique used to separate
non-volatile mixtures. Thin layer chromatography is performed on a sheet of glass,
plastic, or aluminium foil, which is coated with a thin layer of absorbent material,
usually silica gel, aluminium oxide, or cellulose. This layer of absorbent is known as
the stationary phase.

After the sample has been applied on the plate, a solvent mixture (known as the
mobile phase) is drawn up the plate via capillary action. Because different analytes
ascend the TLC plate at different rates, separation is achieved. Thin layer
chromatography can be used to monitor the progress of a reaction, identify
compounds present in each mixture and determine the purity of a substance.
Specific examples of these applications include analysing ceramides and
fatty acids, detection of pesticides or insecticides in food and water, analysing
the dye composition of fibres in plants and their constituents. Several enhancements
can be made to the original method to automate the different steps, to increase the
resolution achieved with TLC and to allow more accurate quantitative analysis. This
method is referred to as HPTLC or ‘’high-performance TLC’’ (Harry and
chrisopher,1989).

We used this TLC to identify the standard sugars such as lactose, xylose, fructose,
glucose, galactose, sucrose, and suitable mixtures of unknown sugars.
Sucrose is a crystalline disaccharide of fructose and glucose (C 12H22O11),
found in many plants but extracted as ordinary sugar mainly from sugar cane
and sugar beets, widely used as a sweetener or preservative and in the
manufacture of plastics and soaps also called saccharide. Glucose is the main type
of sugar in the blood and is the major source of energy for the body’s cells. Glucose
comes from the foods we eat or the body can make it from other substances.
Glucose is carried to the cells through the bloodstream. Several hormones, including
insulin, control glucose levels in the blood. Lactose is a sugar present in in milk. It is
a disaccharide containing glucose and galactose units. Galactose is a sugar of the
hexose class which is a constituent of lactose and many polysaccharides. Xylose is
a sugar of the pentose class which occurs widely in plants, especially as a
component of hemicelluloses. Ethyl acetate colourless volatile flammable liquid,
CH3COOC2H5, used in perfumes, flavourings, lacquers, pharmaceuticals, and
rayon and as a general solvent (Reich, Schibli,2007).

METHOD AND MATERIALS

Deproteinization: 3 ml of ethanol was added to 1 ml of fruit juice, thoroughly mixed,

and denatured protein centrifuged to be removed. Chromatography: the
supernatant was carefully spotted on a thin layer plate together with some standard
sugar solutions. The plate was placed in a chamber saturated with solvent and
chromatogram was developed until the solvent front was close to the top of the plate.
The line was drawn across the plate at the point and the chromatogram was
removed when the solvent reached the mark. The plate was dried in a stream of cold
air and the sugars were located by spraying the plates with freshly prepared aniline-
diphenylamine in a fume chamber and were heated briefly at100°c. The colour of
each sugar was noted with the RF. The RF was used to identify the present sugars
in the fruit juices.

DISCUSSION

Chromatography is a process characterized by the uninterrupted flow of a mixture

(gas or liquid) which, through various means, allows for the differential
migration of the components of the mixture. This definition refers to two
common characteristics of all chromatographic techniques. They are dynamic
processes involving the continual movement if one phase (mobile phase), which
originally contained the mixture, relative to the second phase (stationery phase),
which is immobile and separation is achieved due to differences in the extent to
which individual components in the moving phase interact with the stationery phase.
In identification of sugars in fruit juices we decided to use thin layer chromatography
because it possess the following features: not expensive and time
consuming clean-up of samples is usually necessary, good to excellent solute
resolution and reproducibility of results, obtained quickly (0.2-3 hours), minute
amounts (micro-grams) of material can be detected, inexpensive and easy to use,
excellent for comparative qualitative analysis (Christopher,1989).

After spraying sugars, we then saw the sugars with their different distances and
colours. The distance of a solvent is 9.7 cm, the first sugar is lactose which is 1.8
cm, followed by xylose 6.3 cm, followed by fructose 4.9 cm, followed by glucose
4.4cm, followed by galactose 3.8cm, sucrose 3.5 cm, three unknown sugars,
unknown(1a) 4.5cm, unknown(1b) 6.5cm, unknown (2a) 3.3cm, unknown(2b) 4.1cm,
unknown(3a) 1.8cm, unknown(3b) 4.8cm, and lastly the fruit juice(1) 3.4cm, fruit
juice(2) 4cm, fruit juice(3) 5cm. Sucrose seems dark brown in colour, galactose is
dark blue in colour, fructose is orange in colour, xylose is blue/green in colour,
glucose is light green in colour and the first mixture had three colours, orange, light
green and a dark brownish colour in colour. Looking at the colours, RF values, and
the distance of the mixtures it is observed that unknown (1a) is glucose, (1b) is
xylose, (2a) sucrose, (2b) galactose, (3a) lactose, (3b) fructose.

CONCLUSION

Finally I can conclude that the experiment was conducted successfully,

because we managed to identify the unknown sugars with help of the given
standard sugars
REFERENCE LIST

1.Vogel, A.I., Tatchell, A.R., Furnis, B.S., Hannaford, A.J., and Smith, B.W.J. (2007).
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2. Fair, J.D., Kormos, C.M. (2008). Chromatogram Oxford University Press.pg. 49-
54.

3. Harry W.L and Christopher J.M (1989), Experimental organic chemistry: Principles
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4. Jork, H., Funk, W., Fischer, S.G and wimmer, H. (1990) biochemistry 5th ed.

5. Hector, M, Chavez, L and Kelly, N. (2007), High-performance thin-layer

chromatography for the analysis of medical plants. New York: Theme. Showing
the thickness value of commercial regular and preparative thin layer chromatography
plates