HPLC Retention Time

[Farzana Baksh]

Hello,
I have a question about HPLC. In reverse phase the mobile phase is polar and the stationary phase is non polar. Therefore the most polar component of a sample is eluted from the column first. I understand this statement. However I don't understand why increasing the mobile phase polarity increase the retention time of the components. If the mobile phase polarity is increase then the polar solute with travel faster resulting in decreased retention time. These statements are in my textbook and i feel that they contradict. Is there something more that is going on in the stationary phase? Someone please help. Thank you.

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[Uwe Neue]

"If the mobile phase polarity is increase then the polar solute with travel faster resulting in decreased retention time."

This statement is wrong (if it is meant ot describe RPLC). Exactly the opposite is happening. Get a better textbook!

The key is the partitioning of a solute between the polar solvent and the non-polar stationary phase in reversed-phase chromatography. The solute will stick to the stationary phase, if it is more non-polar. It will stick less to the stationary phase if it is more polar. You can change its retention by changing the polarity of the mobile phase (if you want, the mobile phase is competing with the solute). Thus a more polar mobile phase will increase retention, and a more non-polar mobile phase will decrease retention.

[Farzana Baksh]

Thanks for your response.
But what i dont understand is why increasing the polarity of the mobile phase increases the retention. why would a more polar mobile phase will increase retention, and a more non-polar mobile phase will decrease retentionwhat do you mean by the mobile phase is competing with the solute. Can you give me examples. Thanks alot

[unmgvar]

you need to understand the fact that for most compound polarity is a question of comparison

how polar is the compound compare to:
the mobile phase
to the stationary phase

it is correct if the compound is so polar then it does not matter in a RP column it simply goes out no matter what like uracil

but for other cases it depends,
then the compound is traveling between the stationary phase and the mobile phase. if the mobile phase is much more polar then your compound which is after all organic by nature will stay longer in the stationary phase. hence the longer retention.

[tom jupille]

Think of it this way: "like dissolves like and like sticks to like", and it's "non-polarity" that matters.

Therefore:

For a given column packing and solvent: the more non-polar the analyte, the more tightly it will stick to the non-polar column packing and the longer it will take to come off.

For a given analyte and column packing, the more non-polar the solvent, the better the (non-polar) analyte will dissolve in it and the more quickly it will come off.

For a given analyte and solvent, the more non-polar the column packing, the more tightly the (non-polar) analyte will stick to it and the longer it will take to come off.

[a4analyst]

Hi,

I'm new at this forum and I came across this a bit of old topic. However I was a bit confused with the various conclusions that were stated above. The RPC is a chromatography where we have:
- non-polar stationary phase,
- polar or non-polar analyte,
- polar mobile phase.

Let us discuss two different cases of analyts:
1. polar analyte- due to its polarity it will tend to stay longer in the polar mobile phase than to the non-polar stationary phase. Therefore it is expected that it will elute quite fast, and with the further increase of the polar mobile phase its retention time will decrease (thus the analyte will elute faster).

2. non-polar analyte- the analyte will tend to stick to the non-polar stationary phase therefore will elute slower than the case 1. Therefore this analyte will have longer retention time. In this case if you increase the polarity of the mobile phase (use a more polar mobile phase) then the retention time of the analyte will increase even more (thus the analyte will tend to stay stick to the stationary phase).

I hope that i've cleared some of the things mentioned before. Feel free to correct me if I'm wrong.

[MSCHemist]

In reverse phase everything is still water soluble. The mobile phase typically starts with mostly water and some organic (still polar and water soluble) solvent typically Methanol or Acetonitrile. Methanol is still polar due to its alcohol group though not as polar as water. Acetonitrile is polar as well due to its nitrile functional group though it is even less polar than methanol (no polar hydrogen not an h-bond donor). The amount of the less polar organic solvent and how polar the analyte is determines when it is eluted. Analytes that are less polar tend to stick to the bonded phase more and elute later.

[HW Mueller]

a4analyst, your first conclusion/summary is wrong. If a polar compound is retained, even if only slightly, it will be retained more if the polarity of the mobile phase is increased.

[a4analyst]

HW Mueller it would be nice if you could explain your statement, because there are few people up that have stated the same but no one has baked up their statements with explanation or facts.

[HW Mueller]

First of all just try it out, secondly it is obvious as there is less adsorbant with which to compete at a lower org. content, thirdly check on why some people use RP columns that can be run at 100% water.
(Of course, the "obvious" statement is not so obvious to someone who doesn´t know that,, barring the case of solubility and adsorbance extremes, the adsorbance on the stationary phase is more important than what is going on in the mobile phase.)

[a4analyst]

Ok Mueller I thought about my upper statement and I can see where my mistake was made and why. I thought only of the polarity of the mobile phase not considering its STRENGTH. Considering the polarity of the solvents they go H2O > MeOH > ACN, however considering their strength they go ACN > MeOH > H2O. Therefore when increasing the polarity of the mobile phase by increasing for example the % of H2O you decrease the strength of the mobile phase and that is why your analyte will have increased Rt.

P.S. Thank you for correcting me, however there is no need for your POMPOUS speech regarding the fact that you don't have a clue about the quantity of my knowledge.