Protein seperation - HPLC

9 important questions on Protein seperation - HPLC

Why is there pressure in the HPLC?

Otherwise the process goes to slow.

How does a column based on size work.

There is a solvent with + charge in the colum and the proteins with the – chars keep stuck and the ones with the + charge wand to go out as fast as possible.

How does a column based on size work.

There is a solvent that makes a network and the small particles are stuck on the upside of them and the larger particles fall trough it.
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How does a column based on Affinity work?

Protein binds to ligand and because of that sticks in the column will the rest is flushed away.

What are the aplications of HPLC?

Seperation
Identification
Purificaton
Quantification

How does the detector detect a protein?

Proteins have amino acids that absorb light between 260 and 290 nm. If there is this absorbtion you know that there is a protein.
Or by ultraviolet absorbiton
fluorescence (if the proteins are labeld)
Redioactivity (if the protiens are labled)

Electrochemistry: if the portiens underwent an oxidation or reduction reaction)

What does MuDPIT stand for?

Multidimentional protein identification technique.

How do the Colums let go of the proteins?

By slowly increasing the salt concentration.

What is the benefit of MuDPIT?

You can analyse samples with a lot of different proteins.

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