Describe the laboratory separation techniques used in the separation of amino acids and proteins (10)

Question 1

Chromatography

Answer 1

Is used to separate amino acids based on their solubility in the solvent and their affinity to the stationary phase. In paper chromatography, amino acids are spotted onto the stationary phase and a solvent travels through the paper. The different Rf values (ratio of distance travelled by the compound to the solvent) help distinguish amino acids based on their polarity.

Question 2

Gel electrophoresis

Answer 2

Separates proteins based on their charge and size when subjected to an electric field. Proteins are placed in a gel matrix and an electric current is applied. Proteins with different net charges migrate at different rates towards the electrode of opposite charge. Small proteins travel further through the gel. SDS-PAGE is a common variant where proteins are denatured and given a uniform charge, allowing separation solely based on molecular weight.

Question 3

Isoelectric Focusing

Answer 3

Separates proteins by their isoelectric point , the pH at which they have no net charge. A pH gradient is established in a gel, and proteins migrate until they reach the region matching their Isoelectric point. This technique provides high-resolution separation because proteins stop moving when they are electrically neutral, leading to sharp protein bands.

Question 4

In affinity chromatography

Answer 4

Proteins are separated based on their specific binding affinities to ligands immobilized on a matrix. For example, antibodies or enzyme substrates may be used as ligands to capture specific proteins. The protein of interest binds to the ligand, while other proteins are washed away. The bound protein is then eluted by adding a competitor molecule or changing the conditions. This technique is highly specific and efficient for purifying target proteins from complex mixtures.

Question 5

Ultracentrifugation

Answer 5

uses high-speed centrifugation to separate proteins based on their size and density. Proteins are subjected to gravitational forces, causing heavier (larger) proteins to sediment faster than lighter ones. Density gradients are often used, allowing proteins to migrate until they reach a point where their density matches the gradient, facilitating separation.

Question 6

HPLC

Answer 6

Is a high-resolution technique used to separate proteins and amino acids based on their interactions with a column matrix and their solubility in the mobile phase. The column is packed with fine particles, allowing for high-pressure separation of proteins by size or polarity. HPLC provides precise, quantitative separation and is often used in analytical applications to identify and quantify proteins or amino acids in complex mixtures.