Help on: Mobile phase composition in HPLC

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Question:
"I wish to have a very "elementary" and concise knowledge for reverse and normal phase column chemistry. Articles that I have read so far are very confusing.
Non-polar stationary phase (RP) requires polar mobile phase hence polar compounds come out first (shorter RT) while the non polar compounds come out later...Which component in the MP affects what? e.g. ACN : H2O ...65:35 ...Which one to increase or decrease to effect the chromatogram and in what order would the peaks elute? "

Answer:
“When we use the term reversed phase HPLC we are usually referring to the most common type of HPLC, namely partition. In this technique a non-polar stationary phase, typically an alkyl chain such as C8 or C18, is used together with a polar stationary phase, typically a mixture of water with an organic solvent, such as acetonitrile or methanol.

Consider an analysis in which we wish to analyse a mixture of different organic components which all have some polar characteristics due to the functional groups present. The mixture is injected into the mobile phase and thus introduced to the stationary phase. The organic components in the mixture are attracted to both the non-polar stationary phase and also the mobile phase in which they are dissolved, largely due to hydrophobic forces. When the component is in the mobile phase it moves through the column and when it is in the stationary phase it does not. The equilibrium experienced by each component of interest is a partition between the bonded stationary phase and the liquid mobile phase. The mixture can be separated if the extent of this partition is different for each component.

The relatively more polar components will elute from the column first with the shortest retention times because their partition favours the polar mobile phase. The relatively less polar components will elute from the column last with the longest retention times because their partition favours the non-polar stationary phase and thus they spend more time there during the analysis.

Now to move on to the question of the effect of the composition of the mobile phase, the polarity of the mobile phase can be adjusted by changing the proportions of the aqueous and organic solvents present. This has the effect of either increasing or decreasing the retention times for all the components in the mixture. As the organic portion of the mobile phase is increased the retention times will decrease. This is because the organic solvent attracts the components into the mobile phase and thus they spend less time on the stationary phase. This is why we steadily increase the proportion of the organic solvent in gradient HPLC. There is a rule that you can use to predict the effect of changing the composition of the mobile phase which applies to most small molecules in reversed phase conditions:

‘An increase of the organic solvent component in the mobile phase of approximately 10% will result in the retention time being reduced by a factor of 3.’

So for your example, if we change the proportions of the mobile phase ACN: H2O, 65:35 to ACN:H2O, 75:25 we would expect the retention times of your components to be decreased by a factor of 3.

If you change the proportions of the mobile phase for an isocratic method (as in the example above) then the elution order of your peaks should remain the same. However if you change the mobile phase proportions during an injection (a gradient analysis) you may see changes in the order of elution of the peaks, an effect that is used in the method development process.

Normal phase partition HPLC works in much the same way as described above except that the stationary phase is polar and the mobile phase is non-polar and therefore the elution order is reversed. However, the rule of 3 does not apply. It is important to realise that another type of normal phase HPLC, adsorption HPLC (in which the stationary phase is typically bare silica), does not work on the partition principle but rather the analytes and the mobile phase compete for adsorption sites on the surface of the stationary phase. Normal phase partition HPLC (using polar bonded phases) is most suitable for polar components whereas normal phase adsorption HPLC is most suitable for non-polar components.”
 
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