Chromotography🩸practice

Question 1

What is chromatography?

Answer 1

Chromatography is used to separate substances and provide information to help identify them.

Question 2

Explain the method of chromatography?

Answer 2

1-Take chromatography paper and use pencil to draw 1cm line from the bottom of paper.
2-use pencil to dot 5 point or crosses evenly along the crosses label them a,b,c,d,e
3-take beakers with capillaries test tube and add a single dot of ink in crosses holding for 2-3 seconds.
4-full beaker with solvent(water) of 100ml and take the paper with a clip and stick place it in the beaker.
5-if water is much it little use pipette to addd or remove water so it won’t plush.
6-wait for the components of ink samples to diffuse up the chromatography paper.
7-take the chromatography paper out before it reaches the top of paper
8-let it dry for 10 minutes
9-calculate the retention factor. Distance of Solvent/distance travelled by substance

Question 3

Explain why different pigments in chromatography paper moved up the paper at different rates by including solubility and size of pigment.

Answer 3

Solubility:More soluble pigments will dissolve more readily in the solvent, enabling them to move more quickly up the paper.Those less soluble in the solvent will have a slower rate of movement.

Size of Pigment Molecules: Larger pigment molecules tend to move more slowly through the chromatography paper,

Question 4

Suggest whether proteins are suitable for chromatography?

Answer 4

Traditional one it won’t work
But with

Gel Filtration (Size Exclusion Chromatography)

Ion Exchange Chromatography

Affinity chromatography

It will work for better purification and separation.

Question 5

Suggest how chromatography can be used to identify the amino acids in an unknown protein

Answer 5

Hydrolysis of the Protein: The unknown protein is hydrolyzed into its constituent amino acids using acid hydrolysis (commonly using hydrochloric acid) or enzymatic digestion.

Derivatization: The resulting amino acids is derivatized (adding specific reagents to the amino acids, creating new compounds)to make them suitable for chromatographic analysis. This process often involves adding a compound that reacts with amino acids.

Chromatographic Separation: The derivatized amino acids are then separated using a high-performance liquid chromatography (HPLC) system equipped with a column specifically designed for amino acid separation.

Detection and Identification: As the separated amino acid derivatives wash out from the column, they are detected using methods such as UV-Vis spectrophotometry or
fluorescence detection.
By comparing retention times and peaks to known standards, the individual amino acids in the unknown protein can be identified.

Question 6

What is the separation of amino acids depend on?

Answer 6

size, charge, and affinity to the stationary phase.