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

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

a) Loss of volatile constituents b) Loss of water c) Crystal growthd) Absorption of watere) Polymorphismf) 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 metals3. Organic peroxides promote chain initiation and propagation

DRUGS WHICH DECOMPOSE BY OXIDATIVE PATHWAYS:

Eg : arachis oil, ethyl oleate, riboflavin, vitamin A.

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 development2. Conversion of ergosterol to vit.D.

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

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 = 3.4cmB- Ascorbic acid + sodium hydroxide = 3.6cm