Chromatography Forum

For reverse phase chromatography why is acetonitrile and methanol used instead of just pure water?

For reverse phase chromatography why is acetonitrile and methanol used instead of just pure water?

ACN and methanol are the common organic solvents needed to separate components with reverse phase chromatography. ACN is considered somewhat more toxic than methanol, but ACN produces lower column pressures than methanol.

I'll bet someone with a PhD can explain more in-depth.

In LC there is a strong solvent and a weak solvent. For RPLC, the strong solvent is organic, typically MeCN or MeOH. There are papers out now looking at alternative and 'greener' solvents like propylene carbonate and ethyl lactate. Before MeCN and MeOH came to dominate RPLC, other solvents like glyme, THF and acetone were used.

Issues with solvents include the viscosity, which will dictate the backpressure on the pump and efficiency of the separation, as well as the UV cut since UV-Vis became the most common detector.

The quick pitch; MeCN has a low viscosity allowing for faster flow rates and quicker separations, in addition a UV cut of 190 nm allows for the detection of compounds with weak chromophores.

[Yes, my degree was in this area.]

But how do the organic modifiers actually work. Is it that water on its own is too polar and will leave some compounds on the column unless you mix in some organic modifier in it? I think it is due to polarity the organic modifiers are used but not sure of the chemistry behind what happens a compound that just has loads of water to attach to..what would make a compound that is dissolved in only water want to stay in the column and not elute?

Consider that the organic molecules "like" the C18 molecules better than they "like" water.

You have to think of the chromatographic process as a dynamic equilibrium.

In each small section of the column (also referred to as a "plate" in an analogy to distillation) there is a distribution equilibrium of the analytes between the mobile and the stationary phases.

Fresh mobile phase flows into this "plate" and then again a distribution equilibrium is established. The constant repetition of this process causes the migration of the individual substances.

You can imagine that the equilibrium of distribution for a hydrophic substance will be mostly (99.99% on the side of the hydrophobic stationary phase and not water. So the substance won't reach the end of the column in finite time. The equilibrium of the substance between methanol/acetonitrile and C18 is on the side of the solvent, so it will elute very fast. To get a separation, you need to balance the polarity of the mobile phase by mixing water and solvent.

You can do RP-HPLC with pure water if you have very polar analytes (and a column that can tolerate pure water).

"You can imagine that the equilibrium of distribution for a hydrophic substance will be mostly"

Did you mean to say hydrophobic here?

Sure, sorry for the typo.