This document describes testing the degradation of ascorbic acid using thin layer chromatography (TLC). It discusses the principles of chromatography and TLC. The procedure involves spotting ascorbic acid solutions with and without sodium hydroxide on a TLC plate coated with silica gel as the stationary phase. The mobile phase moves up the plate by capillary action. The Rf values are calculated and show the degraded sample moves slightly farther, indicating degradation.

1. TESTING THE DEGRADATION OF COMPOUNDS USING TLC FOR
ASCORBIC ACID
AIM: To test the degradation of compounds using TLC for ascorbic acid.
REQUIREMENTS: Silica gel, TLC plates, Sodium hydroxide, Ascorbic acid, UV
light or iodine chamber.
PRINCIPLE:
1) CHROMATOGRAPHY: Chromatography is a non-destructive procedure for
resolving a multi component mixture of trace, minor, or major constituents into its
individual fractions. Different variations may be applied to solids, liquids and
gases.
TLC: TLC is similar to paper chromatography except that a thin (0.25mm) layer of
some inert material such as AL2O3, Mgo or SIO2 is used as substrate instead of
paper. The principle of separation is adsorption. One or more components are
spotted on a thin layer of adsorbent coated on a chromatographic plate.
The principle of separation is adsorption. One or more
components are spotted on a thin layer of adsorbent coated on a chromatographic
plate.
2) The components are separated on a thin layer chromatographic plate based on
the affinity of the components towards the stationary phase.
3) The mobile phase solvent flows through because of capillary action (against
gravitational force)
4) The component with more affinity towards the stationary phase travels slower,
the component with lesser affinity towards the stationary phase travels faster.
STATIONARY PHASE: In TLC a thin layer (0.25) of some inert material such
asAL2O3,Mgo,Sio2 is used as a substrate. A layer of any one of these oxides is
made from slurry of powder in a suitable inert solvent. The slurry is spread evenly
over a flat surface (glass) and dried. It may be spread manually or mechanically.
The advantage of using inert substrates instead of paper is that more reactive

2. developing reagents, such as strong acids can be used to detect the compounds
without destroying the substrate.
MOBILE PHASE: Mobile phase used in solvents of different polarity. Stationary
phase i.e. silica contains some free S-OH groups. These groups from H-bonds or
vanderwaal interactions with the analytic components thus adsorption takes place.
The Retention factor[Rf] is determined, which is specific for a compound and it is
calculated by a formula
Distance travelled by the solute
Rf =
Distance travelled by the solvent front
Drug degradation occurs during storage because of chemical alteration of active
ingredients or additives.
 Solubility studies are necessary for following reasons:
1) Product instability or chemical degradation of active drug may lead to under
medication due to the lowering of the concentration of the drug in dosage form.
Example: Digoxin and theophylline
2) Drug decomposition of active drug toxic products may be necessary.
Example: P-amino salicylic acid is converted into P-amino phenol which is
toxic.
3) Instability due to change in physical appearance.
Example: Mottling of tablets, creaming of emulsions.
TYPES OF DEGRADATION OF PHARMACEUTICAL PRODUCTS:
1) Physical degradation or decomposition of drugs
2) Chemical degradation or decomposition of drugs

3. a) Loss of volatile constituents
b) Loss of water
c) Crystal growth
d) Absorption of water
e) Polymorphism
f) Color changes
CHEMICAL DEGRADATION OF DRUGS:
HYDROLYSIS: Drugs with ester and amide group react with one molecule of
water and undergo hydrolysis. Ester groups breaks faster than amides.
Few drugs which decompose by this are:
Aspirin and procaine - esters
Chloramphenicol and ampicillin –amides
OXIDATION: A substance is said to be oxidized if electrons are removed from
it. Thus,a substance is oxidized when it gains electronegative atoms or radical or
loses electropositive atoms or radical’s oxidation often involves the addition of
oxygen or the removal of hydrogen. The simplest type of oxidation is the
elimination of an electron.
The reaction between the compounds and molecular oxygen is called
autoxidation.
The general principle that govern an oxidation reaction:
1. Presence of atmospheric oxygen
2. Presence of trace metals
3. Organic peroxides promote chain initiation and propagation
DRUGS WHICH DECOMPOSE BY OXIDATIVE PATHWAYS:
Eg : arachis oil, ethyl oleate, riboflavin, vitamin A.

4. AUTO OXIDATION KINETICS OF ASCORBIC ACID:
INFLUENCE OF METALS:
Ascorbate ion insolution Cu2+ semiquinone 02 dehydroascorbicacid
Slow oxidation rapid oxidation
INFLUENCE OF AIR ON OXIDATION:
Ascorbic acid reacts with oxygen and then depletes free o2 , when air is bubbled
through the reaction mixture, the rate of oxidation enhanced.
INFLUENCE OF ACIDIC AND BASIC ION:
H+, OH-Ascorbic
acid dehydro ascorbic acid ketogluconic acid
Threonic acid+ oxalic acid
In general, oxidation proceeds more readily in alkaline than in acidic media.
PHOTOLYSIS: decomposition of pharmaceutical compounds resulting from the
absorption of radiant energy in the form of light. The radiations absorbed from the
UV and violet portions of the light spectrum are more active in initiating chemical
reactions than those absorbed from longer wavelength portion of the spectrum.
Drugs which undergo light induced chemical degradation are photo
labile drugs.
FEW PHOTOCHEMICAL DEGRADATION REACTIONS:
1. Color fading / color development
2. Conversion of ergosterol to vit.D.

5. PROCEDURE: prepare mobile phase ( n-butanol : acetic acid : water) (6:2:2)
parts. The mobile phase of about 20ml is to be taken in a beaker, and allow the
beaker to saturate for some time by closing it with aluminum foil. Than, take the
TLC plate which is of 1cm length and 3cm width and then draw line of 1cm on
plate. Prepare two solutions i.e (ascorbic acid and water) and (ascorbic acid and
0.1N sodium hydroxide) and place the two spots on the plate and allow the mobile
phase to run through the plate, it is an ascending type of chromatography. Then
take the plate and dry it observed it under UV light or iodine chamber and calculate
the Rf value.
REPORT: The Rf value for pure ascorbic acid with water and ascorbic acid 0.1N
sodium hydroxide solution were found to be 0.64 and 0.67 respectively.
The degraded ascorbic acid Rf value was increased slightly than the pure ascorbic
acid

6. OBSERVATIONS:
Distance travelled by the solvent = 5.3
Distance travelled by the ascorbic acid + water =3.4
Distance travelled by ascorbic acid + sodium hydroxide =3.6
Rf value of pure drug = 3.4/5.3= 0.64
Rf value of ascorbic acid + sodium hydroxide=3.6/5.3= 0.67
A B
Where
A- Ascorbic acid + water = 3.4cm
B- Ascorbic acid + sodium hydroxide = 3.6cm