HPLC, Reverse Phase HPLC, and ELISA

[Radioactive1112]

Hey,

I need to make sure I understand these techniques can someone help me.

HPLC (normal) = the mobile phase runs through the stationary phase and if the compound is more like the stationary phase it will take longer to elute and high a higher retention peak? When I say higher I mean farther down the graph. and if the substance is more like the mobile phase it will elute quickly.

Is it normally the stationary is polar and mobile non polar.

In reverse phase the stationary is non-polar and mobile polar??

ELISA- I always get questions on ELISA wrong. I guess I just dont understand the technique any simple explanations?

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[NextStepTutor_1]

Your understanding is correct. Typically the plate (stationary phase) is polar and the mobile phase is non-polar. This is for normal HPLC and is similar to thin-layer C or column C. As the non polar solvent is sent through the polar plate, non polar compounds will be attractive to the solvent and migrate through the unattractive plate, while polar compounds will bind more favorably to the plate and migrate less. Non polar compounds in this example will have a faster elution time, higher rf value, and lower retention.

Reverse-phase HPLC is when the phases are reversed, obviously. But this means the mobile phase is now polar and the stationary phase is non polar.

Check out this link if HPLC is still confusing:
http://www.chemguide.co.uk/analysis/chromatography/hplc.html

ELISA is used to identify the presence and quantity of a particular protein. This site could help:
http://www.bio.davidson.edu/genomics/method/ELISA.html

 

[Radioactive1112]

Life saver! Thank you very much! I love it when things click

 

[MedicineN'Jazz]

A great acronym I like to use for TLC and HPLC = "polar is lower and slower."
"Lower" indicative of the Rf value in comparison to the non-polar molecules, and "slower" as a representative of the slow(er) retention time. Hope this helps!

 

[aldol16]

Is it normally the stationary is polar and mobile non polar.

In reverse phase the stationary is non-polar and mobile polar??

The reason we use both is because sometimes, when you're trying to do a tough separation, your compound is too polar and just sticks onto the column and never comes off. This is a problem if that is the compound you're trying to isolate. So if you then switch it up to reverse phase, your compound of interest will elute first.

 

[DSM_302.0]

A great acronym I like to use for TLC and HPLC = "polar is lower and slower."
"Lower" indicative of the Rf value in comparison to the non-polar molecules, and "slower" as a representative of the slow(er) retention time. Hope this helps!

Is it the case that for both HPLC & for RP-HPLC that polar is always lower and slower?

 

[DSM_302.0]

I was confused by this because I've never seen HPLC and was imagining it to be like TLC, and 'eluting' first to mean 'stick to the slide first'. But no! It's a column and 'eluting first' means to come out the other end first.

This video helped for anyone else searching for AAMC Sample Test C/P 18:

 

[aldol16]

Is it the case that for both HPLC & for RP-HPLC that polar is always lower and slower?

Reverse-phase reverses everything you would expect from normal phase. Think about how they work. Normal phase you start with a polar stationary phase and a nonpolar mobile phase. So the polar stuff will stick and thus have a longer retention time. Reverse-phase starts with a polar mobile phase (usually water with a C18 column) and a nonpolar stationary phase (usually C18 column). So the nonpolar stuff will "stick" and the polar stuff will wash out first.

 

[MedicineN'Jazz]

Is it the case that for both HPLC & for RP-HPLC that polar is always lower and slower?

Nope, only HPLC, TLC and paper chromatography. So for RP-HPLC, polar is "higher and faster"