Digitalis Tlc

Date:

-09-09

SEPERATION AND IDENTIFICATION OF DIGITALIS GLYCOSIDES BY TLC. AIM: To carry out the TLC of the given sample of drug (Digitalis powder). REQUIREMENTS: Beakers, round bottom flask, TLC plate, hot air oven, 5% w/v potassium hydroxide solution, silica gel G, distilled water, methanol iso-propanol , ethyl acetate, Dichloromethan , heating mantle, stirrers, conical flask, pipette, measuring cylinder, mortar pestle, funnel, filter paper. REFERENCE: (1) “Herbal Drug Technology”, by S.S. Aggarwal, M. Pandhavi, universities press, pg no 231, 258-262; (2) “Plant Drug Analysis- a thin layer chromatography atlas”, 2 nd edition by H. Wagner, pg no 99-113; (3) “Quality Control of Herbal Drugs- an approach to evaluation of botanicals” by Dr. Pulok mukherji, pg no 426-482. THEORY: Introduction: chromatography is a broad science of physical method meant to separate and analyse complex molecular mixtures. It depends on the differential affinities of the solutes in two immiscible phases, a fixed bed with a large surface area of a fluid which moves through or over the surface of the fixed on stationary phase. TLC is a method of analysis in which the stationary phase, a finely divided solid is spread as a thin layer on a rigid supporting plate and the mobile phase, a liquid, is allowed to migrate across the surface of the plate by capillary action. Adsorbents for TLC: various types of tested adsorbents are available for TLC. They differ from the usual materials in that their structure is fine graded i.e. finely divided, with the grain size of the adsorbent lying between 5 to 50 and passing through a number 200 screen. (1) Silica gel: it is the most extensively used adsorbent. It is employed as such for adsorbent TLC and muclified for reverse phase chromatography using substance such as octadecylsilyl to it. It is suitable for constituents like amino acids, alkaloids, sugars, fatty acids, lipids, essential oils, steroids and terpenoids. Page | 1

Date:

-09-09

(2) Alumina: it has basic surface and is chosen over silica gel for

separation of weakly polar compounds. Similar to silica gel alumina can be obtained in various forms like alumina and alumina H1 and alumina F 254 etc. It is suitable for alkaloids, food dyes, phenols, steroids, vitamins, carotenoids and amino acids. (3) Kieselguhr: it is a natural adsorbent with low. It is not used to a very large extent. Other inorganic adsorbents are CaSO4, magnesium silicate, magnesium oxide, bentonite etc. Kieselguhr is suitable for sugars, dibasic acids, fatty acids, triglycerides, amino acids and steroids. Organic adsorbents: cellulose and its derivatives are used exclusively for separation of hydrophobic compounds like amino acids, nucleic acids, carbohydrates and closely related isomers. It is suitable for amino acids, food dyes and alkaloids. Polyamides (nylon): nylon is a long chain polymer which because of the presence of many free amide of carboxylic on its surface is an adsorbent with strong hydrogen bonding abilities. It is suitable for anthocyanins, antioxidants, flavonoids and proteins. Solvent system: the solvent system is chosen by the trial and error method. The rate of migration of a compound depends on the solvent used. The simplest systems are mixtures of organic solvent used to separate mono and bi-functional molecules by adsorptive chromatography on layers of activated silica gel or alumina. Solvents at the bottom of the series are polar and move most of the compounds where as those at the top are non-polar and move few compounds. If the chemical nature of the solute to be separated is known, a suitable solvent can be selected using stahl’s triangle. Selection of supporting plate: the carrier on supporting plate is the backbone of the entire chromatographic apparatus, and hence it must be stable in presence of all types of solvents, reactive spray reagents, and also at high temperatures. Glass plates fulfill these requirements best. Their thickness is usually chosen between 1.8mm to 5.5mm. Borosilicate glass is also used when a reaction is carried out at very high temperature. Preparation of thin uniform layer: the film thickness varies from 0.2mm generally 0.22mm thickness is used. Page | 2

Date:

-09-09

Thin layer can be prepared by the following methods: 1. Dipping: plates are dipped into a slurry of the stationary phase suspended in a volatile solvent. The solvent is then removed by air drying or by heating the plates in an oven. The development time for this method is very short. 2. Spraying: an aerosol spray is used to prepare thin layer, using a slurry of suitable consistency. 3. Pouring: a known amount of slurry is poured onto the plate. The plate is then tipped back and forth to spread slurry. 4. Spreading: an application as spreading is used and the layer thickness can varied as desired. Sample preparation and application: the dried are conditioned if necessary, in a controlled humidity chamber. Samples ranging from a few micrograms are dissolved in 10-100ml of volatile solvent. They are then applied as apots or as thin streaks with a capillary tube or a micro line syringe. The solvent maybe evaporated between successive applications. Area of sample application should be as small as possible. Development:--linear --radial --extended --gradient --temperature controlled Chromato plates are placed inclined at about 45 degree in the tank. The tank is closed with a lid and the solvent is allowed to move up the plate from the point of application. When the mobile phase has traveled two-third of the plate, the plate is removed from the chamber, the solvent front is marked and allowed to dry. The various development techniques are: 1. Linear development: the TLC plates are developed linearly in the

ascending, descending or horizontal mode. In ascending mode, the sample is spotted at one end of the plate and then developed in the ascending direction. It is known as vertical development. In the descending mode the solvent is allowed to flow from the reservoir to the plate through a strip of filter paper. It has no advantage in terms of efficiency of separation and speed analysis. Page | 3

Date:

-09-09

2. Radial development: it is also known as circular TLC. It is performed

on horizontal chromate plates. 3. Extended development: it consists of the following techniques: Continuous development technique: it is used to achieve complete separation of compounds which resolve with small differences in Rf value. Multiple development technique: in this repeated development is carried out with the same solvent in the same direction, each time after drying. Two-dimensional technique: it is used to separate complex mixtures. The plates are developed in one axis first and then, after drying they are developed in the other axis with the same solvent or different solvent. 4. Gradient development: in this two different adsorbents are applied simultaneously. Eg: temperature controlled development, in this development is carried out at definite temperature. Detection: before starting the procedure to detect various solutes, the chromatogram is allowed to dry. This is done either at room temperature or in an oven. Application of visualizing agent to help in detection is done in a number of ways - exposure of the chromatogram to vapours - dipping the plate in reagent solution - spraying or impregnating the plate with reagent - allowing the solution to develop as in the normal TLC The visualization procedures maybe classified, for convenience into following: 1. Non-destructive method: UV method for fluoroscent compounds under UV chamber at 254 & 365nm. Iodine vapour treatment also widely used wherein some phyto constituents show brown, amber or yellow zones after exposure to iodine vapour. 2. Reagents causing irreversible reaction: charring is widely used technique for the detection of carbon containing compounds. This involves spraying the plates with sulphuric acid and then heating in an oven at 110 degrees for 10-30mins. The organic compounds are destroyed and a dark point of carbon remains. Page | 4

Date:

-09-09

Analysis: in TLC, qualitative analysis of unknown compound is done by comparing Rf values with standard values. As solutes should never travel the full length of the stationary phase in TLC all the Rf values are less than 1. The Rf value depends on the amount of stationary phase, the humidity, layer thickness, solvent quality, saturation of the chamber, development distance, temperature, amount of substance added and presence of impurities. Rf =distance from origin to point of maximum intensity Distance from origin to solvent front. Rf = retention time PROCEDURE: (a) Preparation of Extracts: Weighed 2gm (>1% total cardenolides) or 10gm (<0.1% total cardenolides) of powder drug.  Extracted the drug by heating with 30ml 50% ethanol and 10ml 10% lead acetate solution under reflux for 15 minutes.  Cool and filtered the extract and collected the filterate.  Filterate was extracted with Dichloromethane & Isopropanol (3:2) for 3 times. (Each time 15ml mixture was taken).[note:-care should be taken to avoid emulsion formation by gental shaking].  Combined the lower phase after sepration and is filtered over anhydrous sodium sulphate.  Evaporated the filterate to dryness and collected the residue after evaporation.  Residue was dissolved in 1ml Dicholoromethane & Isopropanol mixture (3:2) and used for chromatographic sampling. (b) Preparation of standard Solution:-

 5mg of Digoxin was dissolved in 2ml methanol. (c) Adsorbents:- Silicagel G (d) Solvent system: Ethylacetate:Methanol:Water (81:11:08) (e) Spraying reagent:- Kadde’s reagent.  Sample solution + Kadde’s reagent blue/violet colour. Page | 5

Date:

-09-09

 Kadde’s reagent (3,5-di nitro benzoic acid in methanol + 5% w/v solution of KOH).

RESULT:

CONCLUSION:

Page | 6