Choice of Organic solvents (mixtures) for HPLC method?

[seamoro]

I understand that polar solvents disolve polar compounds and non polar solvents disolve non polar compounds. So you obviously use a moblie phase that will not crash your sample out of solution. But what i want to know is how determine the choice of solvent for HPLC moblie phase based on its polarities index...why is its polarites index signficant? why mix water with a polar index of 9 with Meoh with a polarity index 5.1? And how do you work out the ratio of the mixture? Could you not use water and MeOH all the time?..why would you change to a solvent with a lower polarity index?
I know i asked alot of questions here....if somebody can reference a website that will explain this with no waffling i would greatly appreicate it.
Thanks in advance

[skunked_once]

I will attempt an explanation for this. The separation of molecules in chromatography is based on the attraction of the molecules to a solid phase in competition with their attraction to the mobile phase. In reverse-phase chromatography, the column surface is non-polar and the mobile phase is polar. Thus methanol or water which are polar solvents are used for the mobile phase. A major factor therefore is the polarity of the molecules being separated. Those which are more non-polar will tend to stick to the column surface while those which are more polar will tend to dissolve in the mobile phase. No if we assume that we are separating a mixture of molecules with a range of polarities from non-polar to polar, we will need a mixture of mobile phases that will cover this range of non-polar to polar. This is done by mixing varying proportions of water and methanol which have different polarities. Polarity indexes give a relative value to the polarity of the solvents used.

[Uwe Neue]

Unless you are doing preparative chromatography, the solubility of the analyte in the mobile phase is of little concern. The choice of an isocratic solvent composition is best solved by running a gradient and calculating the solvent composition at which the analyte is eluting. I have never in my entire life worried the polarity index, but have written book chapters on method development. So you can see how important the polarity index is. You could use water(buffer)-methanol mixtures all the time, but you will loose out on the important tool of solvent selectivity, if you do not use a different solvent such as acetonitrile.

[Alex Buske]

As soon as you get practical examples it is quite easy to see, why mixtures are used.
When I use (polar) water as eluent, the analytes I am working with are so much attracted to the Stationary phase that they will simply stick there and never come out of the column again.
When I use (less polar) pure methanol they are quite happy in the mobile phase and come out of the column straight without any retention. And no retention means also no separation.
So basically I start with methanol and add water (run by run 5%, 10%...) until they they show enough retention. The more water the more retention.
Practically this is done the other way around, starting with water and adding methanol during the run.
Unfortunately some analytes do not separate on a column with methanol / water mixtures. Then one tries ACN/waters mixtures. Or plays with the pH or the temperature. Or mixes water and methanol or ACN.

Alex

[tom jupille]

This link is old and ugly, but it provides a simplified explanation of why we use the solvents we do:
http://www.lcresources.com/resources/getstart/3b01.htm