The following is the chromatogram of an anion exchange column. The absorbance, v
ID: 490544 • Letter: T
Question
The following is the chromatogram of an anion exchange column. The absorbance, volume of elution, and the salt gradient concentration are shown in the different axes. Two proteins, A and B were purified using the column. Answer the questions based on the chromatogram.
a. Charge of A:
b. Charge of B:
c. Which protein has more net charge to bind to the column tightly?
d. What salt concentration did A elute at?
e. What salt concentration did B elute at?
f. Which of the two proteins are bigger?
Thanks!
-t 0.8 M 0.6 M A 0.4 M 8 0.2M 15 25 35 55 65 85 Volume (mL) Cl uonepuaouoo IOeN B 5 08ZVExplanation / Answer
Ion exchange chromatography is a seperation technique used in the purification of protein. There are 2 types cation exchange and anion exchange chromatography. The seperation is based on the afffinity to the ion exchager. Its usually used when the protein has net charge ie, polar. The stationary phase contains immobile matrix that contains charged functional groups or ligands. In case of cation exchange column the matrix will be negatively charged and retains the positively charged particles via electrostatic interaction and in case of anion exchange column the stationary phase will be positively charged. Here the more the effective charge on the ion , the interaction with the stationaryphase wil be more and elutes last.
a) A will be slightly negatively charged. As it eluted fast the charge will be less.
b) B will be negatively charged. It eluted after A , slowly so it will be having met negative charge more than A.
c) B has more net charge than A and thus has more affiinity to the column as it eluted after A.
d) A eluted at a concentration of about 0.2 M.
e) B eluted at about 0.4M of salt concentration.
f) Its not based on size but mainly on charge. Here the matrix is porous and the larger sized ones have lesser interaction and elutes first compared to smaller ones. So comparatively A might be the one with larger size.
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