ANALYTICAL TECHNIQUES

An analytical technique is a method that is used to determine the concentration of a chemical compound or chemical element in a sample. Example:
Chromatography Autoradiography Electrophoresis

1.Chromatography
Used to separate mixture of chemical substances into its components. Mixtures can be : photosynthesis pigments, proteins, amino acids etc.
This technique involves moving the mixture(normally a suitable solvent), the mobile phase, over a stationary phase (inert/solid matrix such as paper or silica gel)..

Types of Chromatography
Paper Chromatography and Thin Layer Chromatography (TLC)

Column Chromatography

Gas Liquid Chromatography (GLC)

High Performance Liquid Chromatography (HPLC)

4

Paper Chromatography
Mixture of chemicals of similar nature (e.g photosynthesis pigments) is separate by allowing the solvent to flow over them in a solid medium such as paper
Apply a concentrated drop of sample () with a capillary or dropping tube to bottom of paper (origin pencil line) Stand paper in a sealed vessel. carefully add solvent (keep solvent level below sample).

Allow solvent to adsorb up the plate, drawing the sample with it.

and Paper Chromatography

The ratio of distance travelled by the component (from origin) compared with the distance travelled by the solvent front (from origin) is called the Rf value.

x
Solvent front

a
b c

Rf of Rf of

= a/x = b/x

Rf of

= c/x

Paper Chromatography
A solution of a mixture is applied as a spot/band at the bottom of the plate and allowed to travel with the solvent up the plate.
Mixed standards Unknown + standards

standards

A+B+C

A+B+C ?

 Sample for chlorophyll pigment chromatography

Experiment 6

Phaeophytin

2. ELECTROPHORESIS
Is a technique used to separate substances with different charges in an electric Field. Can be use for separating mixture such as Protein/amino acid and nucleic acid.

 The positively charged particles move to the cathode, and negatively charged particles move to the anode. Two factors which affect the speed charged molecules are the amount of charge & size

Gel Electrophoresis
Used to separated DNA fragments by their size.

 The agarose gel provides a matrix with pores to allow molecules to travel through and be sorted by size. The electric current is the force that causes the negatively charged DNA molecules to more toward the positive pole The wells are the "starting gates" for the DNA molecules to be loaded into before starting the "race".

 Different individual will have different band of DNA

Experiment 6

Phaeophytin