By Dodo on Thursday, August 12, 1999 - 11:23 pm:

Hello!
I知 quite new in HPLC and I have some questions. Can anybody explain me, why in RP-HPLC are mainly used mixtures of methanol, acetonitrile or THF as mobile phase? Has anybody heard about using ethanol as mobile phase? What is the problem with other organic solvents? It is only the polarity?

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By M Paciolla on Friday, August 13, 1999 - 05:57 am:

I haven't seen too many methods that incorporate EtOH in the MP for RP-HPLC. However, regarding your question on polarity, the PI for EtOH is 0.88 vs. 4.2, 6.2, 6.6 and 10.2 for THF, ACN, MeOH and H2O respectively. Also, I believe EtOH is more viscous than the other solvents which could lead to high back pressure problems.

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By Tom Jupille on Friday, August 13, 1999 - 08:31 am:

Polarity is part of the story, but there's more.

In a reversed phase chromatography the polarity (or, more properly, non-polarity) of a solvent determines its *strength* (how it affects retention). But that's not a big deal, because mobile phase strength is controlled primarily by blending in water anyway. What's of more interest is the *selectivity* of a solvent (how it affects the relative spacing of the analyte peaks).

Not too surprisingly, the selectivity of chemically similar solvents is similar (i.e., all the alcohols are pretty much alike, all the ethers are pretty much alike, etc.). If you start with an alcohol as your organic solvent, and some of your peaks overlap, it's very likely that you'll see the same overlap if you use a different alcohol. What you want to find is a solvent that is as *different* from an alcohol as possible.

In reversed-phase, you also want miscibility with water (remember, you'll adjust the water content to get the retention about right). The bottom line on all of this is that the three solvents which are the most different from one another and miscible with water are -- you guessed it -- methanol, acetonitrile, and tetrahydrofuran.

This is not to say that other solvents can't be used, or that other solvents may not give better selectivity in specific cases, it's just that these three give the best chance of providing a reasonable separation, so they're the ones we turn to first.

I hope this helps!

-- Tom Jupille / LC Resources Inc.

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By Anonymous on Friday, August 13, 1999 - 12:24 pm:

How about the cost effect? I believe that EtOH will be too expensive to be part of the MP.

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By B.Buglio on Saturday, August 21, 1999 - 02:48 pm:

Two problems with ethanol; (1) Its viscosity can cause high back pressures and (2) its expensive to use ethanol which doesnt contain toluene or water.

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By Anonymous on Tuesday, March 23, 2004 - 10:34 am:

Could anyone please explain what makes the structures of asprin, caffiene and ibroprofen more or less polar..Something to do with the functional groups but im only a very basic biochemist!

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By Anonymous on Tuesday, March 23, 2004 - 04:27 pm:

At the risk of sounding patronizing, go back and find your high-school chemistry textbook. The concept of polarity should have been convered there.

The grossly oversimplified answer is that it has to do with the distribution of electrons in the bonds holding the molecule together. The more "uneven" the distribution, the more polar the molecule. Carbon atoms tend to have even distributions. Oxygen and nitrogen have uneven distributions. Other things being equal, the more oxygen or nitrogen atoms in the molecule, the more polar it is.

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By Bharat Kumar Agarwal on Thursday, May 6, 2004 - 06:05 am:

Can we used a single solvent in HPLC mobile phase,
like water, methanol, acetonitrile ?

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By tom jupille on Thursday, May 6, 2004 - 08:55 am:

Yes, you can, but it probably won't give you a very good separation.

Per another post, go through the "getting started in HPLC" course, then come back and ask questions on the unclear portions:
http://www.lcresources.com/resources/reslinksform.html