EXPERIMENT NO: 08. To study Thin Layer Chromatography (TLC) pattern of isolated Caffeine | T.Y B. PHARM SEM - V PHARMACOGNOSY & PHYTOCHEMISTRY – II

 T.Y B. PHARM SEM - V

PHARMACOGNOSY & PHYTOCHEMISTRY – II

EXPERIMENT NO: 08

AIM: To study Thin Layer Chromatography (TLC) pattern of isolated Caffeine.

REFERENCE:

1.     Dr. Anup K. Chakraborty, Sushma M. Rathod. “Practical in pharmacognosy & phytochemistry – II. Edition 2019, published by S. Vikas & company (Medical publisher), Page no. 36, 50, 51

REQUIREMENTS:

Apparatus: TLC plates, Glass chamber, Iodine chamber, Hot air oven, Beaker, Capillary etc.

Chemical: Isolated caffeine, Chloroform, Ethyl acetate, Methanol, Acetic acid

INTRODUCTION:

Thin layer chromatography (TLC) is mainly used qualitatively for screening of different plant extracts which serve as a very important tool in the phytochemical studies.

Preparation of TLC plate: The glass plates of sizes like 20x20; 20x10; 20x5 cm are used for TLC study. The adsorbents like silica gel or alumina used as a stationary phase. Water is used as a solvent for preparation of slurry. Techniques used for preparation of TLC plates are pouring, dipping or spreading. The prepared TLC plates are air dried and activated in oven at 105°C for 30 min.

Application of samples: Standard borosil glass capillaries are used for applying samples on TLC plates. The spots are applied at suitable distance and allowed to dry.

Preparation of saturated chamber: The TLC plate is placed vertically in a rectangular chromatography tank or chamber. Selection of solvent system depends upon the nature of the phytoconstituent. Saturation of chamber has been done so as to avoid the unequal solvent evaporation losses from developed plate which leads to various types of random behavior and edge effects. The solvent level should be kept below the spots. Generally ascending method is used.

Location of spots: Physical methods include the UV, fluorescence, colour or radioactive counting. In case of chemical methods, locating agents are applied by spraying. Various spraying agents are used such as Iodine, 1% vanillin in sulphuric acid, 50% methanol in sulphuric acid. Total number of spots, their colour, intensity, size and shape observed.

Rf Value:

The Rf value is calculated by using below formula.                             

PROCEDURE:

Preparation of extract: Dissolved 1mg of isolated caffeine in 1ml chloroform or methanol.

Adsorbent (Stationary Phase): Make 15 g silica gel slurry with 20 ml of distilled water. The plates are coated with slurry of silica gel to a width of 0.4mm, allowed to dry at room temperature, activated oven at 120°C for 20 min.

Solvent system (Mobile Phase): The solvent system ethyl acetate-methanol-acetic acid (8:1:1). is poured in the TLC chamber to a depth of 3cm and allow to saturate. The chamber is lined with filter paper on three sides to maintain equilibrium of mobile phase

Application of spots: The above sample applied as two spot on each plate with the help of

Development of chromatogram: The chromatogram is established by ascending technique, allowing solvent system to reach a height of 10 cm. The plates are withdrawn from the chamber, air-dried and visualize the dried TLC plate by exposure to iodine vapour.

Rf Value:

The Rf value is calculated by using below formula

                                         

                                                                                                                                                          

RESULT: The TLC of caffeine was performed and the Rf value of caffeine was found to be…………………….