Unit Number and Title Unit 2, Practical Scientific
Procedures and Techniques
Learning Aim Aim C, Undertake chromatography
to identify components
Student Name Maimunah Ramouche
Assessor Mandeep |Mangat
Undertake chromatography to identify components
Introduction
TLC is a highly versatile separation method that is widely used to analyse a
variety of substance classes, including pesticides, steroids, alkaloids, lipids,
nucleotides, carbohydrates, and fatty acids. In this technique it will be used to
analyse plant pigments and amino acids. Components of a mixture will be
identified using chromatographic techniques and by calculating the retention
factor value, unknown solutions can be identified. Thin layer chromatography
(TLC) uses a TLC plate that contains silica gel and is a method used to check the
purity of liquids; it contains two phases, the mobile and stationary phase.
Aim
Undertake chromatography of plant substances and amino acids
Equipment
- TLC sheets
- Pencil
- Ruler
- Glycerine
- Leusine
- Lysine
- Beaker
- Ninhydrin
- Splint
Safety precautions
Hazard Risk Precaution
Glassware (Capillary Breaking, shards, cuts
tubes, beaker)
Electrical appliances Electric shocks, short Keep dry, unplug when
(Blow dryer) circuiting not in use
Amino acids, Harmful Irritation Gloves, goggles, lab
solvents coats
Ninhydrin Stain skin, irritation to Gloves, goggles, lab
eyes, skin and coat, avoid exposure
respiratory tract
, Propanone Irritation, volatile, Use pipette, goggles,
flammable, vapours avoid skin contact and
open flames
Capillary tubes Glass cuts
Method
Chromatography of amino acids
- A pencil line was drawn 1cm from the bottom of the paper
- A pencil dot was also drawn at 1cm intervals along base line for the amino
acids.
- Used Capillary tubes to apply spots of Amino acids to the line, ensuring
enough sample is spotted on the plate. The dots were labelled.
- Solvent was added to a beaker.
- Folded edge of paper over a splint then placed in beaker, ensuring the
bottom of the paper reached the solvent, but the level was not touching or
above the base line.
- Allowed capillary action to draw the solvent up, then marked the solvent
front.
- Technician sprayed ninhydrin onto the sheet, then dried it.
- This sheet of paper is called a chromatogram and can now be analysed,
and the Rf value of each amino acid spot calculated.
Chromatography of plant pigments
- A pencil line was drawn 1cm from the bottom of the TLC plate
- A pencil dot was also drawn on the base line for sample
- Used pestle and mortar to grind spinach leaves, then added propanone
with a pipette
- Used Capillary tubes to apply a spot of liquid to the line, ensuring enough
sample is spotted on the TLC plate.
- Solvent was added to a beaker.
- Placed plate in beaker, ensuring the bottom of the plate reached the
solvent, but the level was not touching or above the base line.
- Allowed capillary action to draw the solvent up, then marked the solvent
front.
- Rf value of each spot was calculated.
Results
Rf value = Distance travelled by component / Distance travelled by solvent front
