TLC Lab And Protein Extraction

Question 1

Chromatography can be…or..

Answer 1

Preparative(isolate pure form)
Analytical(composition and purity)

Question 2

Components of chromatography

Answer 2

Stationary phase> solid or liquid adsorbed on solid
Mobile phase>liquid or gas
Separated molecules

Question 3

Stationary phase and mobile phase chosen acc to..

Answer 3

Nature if components to be separated

Question 4

Chromatography bed

Answer 4

Column
Planer> TLC/paper

Question 5

Chromatography physical state

Answer 5

Gas
Liquid> HPLC/ supercritical fluid

Question 6

Separation mechanism

Answer 6

Affinity
Ion exchange> anion exchange/ cation exchange
Gel filtration

Question 7

TLC 2 priciples

Answer 7

Adsorption>chemicals retained based on their adsorption. To stationary phase
Partition>separation acc to solubility

Question 8

TLC chamber function

Answer 8

Maintain uniform environment and prevent evaporation of solvents and maintain pressure

Question 9

Filtration paper on chamber?

Answer 9

To keep vapours

Question 10

Stationary phase TLC

Answer 10

Silica gel> covalent structure, very polar, based on polarity
Aluminium oxide> can have different pHs, based on pH
Cellulose

Question 11

Rf calculation

Answer 11

Distance travelled by sample/ total distance travelled by solvent front

Question 12

Larger Rf=

Answer 12

Larger distance travelled, less polarity

Question 13

Separating triglycerides

Answer 13

Stationary phase> plastic coated silica
Mobile phase> petroleum ether 84 :diethyl ether 15 :acetic acid 1
Visualising using iodine or UV

Question 14

Marking baseline with pencil

Answer 14

To prevent scraping the silica
To prevent the ink from interfering with the results

Question 15

Protein solubility

Answer 15

Ratio between hydrophobic and hydrophilic more charged equal more soluble at isoelectric point the net charge is 0

Question 16

Soluble are
Insoluble are

Answer 16

Globular
Fibrous

Question 17

Extraction steps

Answer 17

Cell lysis
Separation of cell debris
Solubilisation of insoluble proteins

Question 18

Cell lysis by

Answer 18

Physical(mortar and pestle/ liquid nitrogen)/ chemical(detergent)

Question 19

Buffer X
Buffer Y

Answer 19

Phosphate buffer+ monosodium phosphate+ disodium phosphate

Sodium dodecyl sulphate + phosphate buffer(solubilise insoluble proteins)

Question 20

Dithiothreitol DTT
Tris
TCEP,
Mercaptoethanol

Answer 20

Reducing agents to inhibit oxidation reactions.

Question 21

Why extract at 4 degrees

Answer 21

To prevent proteases work