Analysing Proteins
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What is thin layer chromatography (TLC) used for in protein analysis? (sprte. + idntfy. AA mtr.)
To separate and identify amino acids in a mixture
What is a TLC plate made of? (smll. sht. gls/plstc./mtl/ ctd. slca. gel)
A small sheet of glass, plastic, or metal coated in silica gel (stationary phase)
Why is silica gel used as the stationary phase? (plr. alwng. sprtn. bsd. plty. AA)
It is polar, allowing separation based on polarity of amino acids
Why must gloves be worn when handling the TLC plate? (prvnt. cntmtn. frm. oils/AA skn.)
To prevent contamination from oils or amino acids on the skin
Why is a pencil line drawn 1.5 cm above the base of the TLC plate? (mrk. orgn. w/o dslvng. slvnt.)
To mark the origin without dissolving in the solvent (pencil is insoluble)
Why is the TLC plate dried in a fume cupboard?
The solvent is toxic and volatile
What happens when the TLC plate is placed in the solvent? (slvnt. trvls. up plt. cryng. AA with it)
The solvent (mobile phase) travels up the plate, carrying amino acids with it
Why must the solvent level be below the pencil line? (prvnt. AA smple. dslvng drctly. slvnt. )
To prevent the amino acid sample dissolving directly into the solvent
How is the amino acid sample applied to the TLC plate? (usng. cplry. tbe. plc. smll. spt. pncl. lne.)
Using a capillary tube to place a small spot on the pencil line
What are the two factors affecting movement of amino acids in TLC? (rttn. SP - slblty. MP)
Retention by the stationary phase Solubility in the mobile phase
How does polarity affect movement in TLC? (>plr. AA - strngr. atrctn. plr. SP - AA mves. lss. but lss. plr. AA - wkr. atrctn. - mvng. frthr.)
More polar amino acids → stronger attraction to polar stationary phase → move less Less polar amino acids → weaker attraction → move further
How does solubility in the mobile phase affect movement? (AA >slble. slvnt. trvl/ frthr. plt.)
Amino acids that are more soluble in the solvent travel further up the plate
Why do amino acids have different Rf values? (diff. plrts. aftng. atrctn. SP + slblty. MP)
Because they have different polarities, affecting their attraction to the stationary phase and solubility in the mobile phase
How is TLC used to identify amino acids? (cmprng. Rf vls. w/ knwn/ stndrds. undr. sme. cndtns.)
By comparing Rf values with known standards under the same conditions
Explain why a more polar amino acid travels a shorter distance on a TLC plate (slc. gl. plr. bng. SP - >plr. AA frm. strngr. intrmlcr. intrctns. w/ SP thn. wll. rtnd. mre.)
The silica gel is polar, being the stationary phase, more polar amino acids form stronger intermolecular interactions with the stationary phase, then will be retained more