Some HPLC questions - beginner

[michi]

Hi,

I'm a beginner in chromatography and I had a practical about it last week. To fully understand the whole process I asked myself some questions and I did some research but some things I still dont understand.

We analysed four compounds in reversed-phase chrom. , acetophenone, nitrobenzene, benzene and toluol using a water: methanol mobile phase.
The first question relates now to the order of elution. Acetophenone was the first to elute, then nitrobenzene, benzene and toluol. I can understand that toluol is last because of its CH3 group present compared to benzene alone, but I dont understand why nitrobenzene comes after acetophenone; nitrobenzene looks more polar in my opinion :S

Can I understand the whole separation system as followed, the analytes are injected and retained by the stationary phase. Depending on the mobile phase, those bonds made with the column are disrupted by forming stronger bonds (H-bonds for example) with the mobile phase.

Whilst doing research I read quite often that if the mobile phase is less polar its solvent strength would be higher. Acetonitrile for example, is slightly less polar than methanol and is therefore a stronger solvant producing smaller retention times. Why is it stronger?

Would it also be possible to separate those compounds using GC?

I really have no clue about those questions, but maybe you can help me to understand a bit more.

michi

[chromatographer1]

Reverse phase chromatography can be quite the 'reverse' of expectations. Remember the SOLUBILITY of the analytes in the solvent is the determining factor of the elution of the analytes, not merely the 'polarity' of the solvent.

"Would it also be possible to separate those compounds using GC? "

Yes. No problem at all. The order depends upon the selectivity of the liquid phase used in the column.

best wishes,

Rod

[Dinky]

Hi,

my first question is; what column did you use? The stationary phase will have an effect on the elution order (as this effects the selectivity of you method).

The most common column to use in Reverse phase HPLC is a C18 column. Different columns have different mechanisms of interactions with the analytes. For example C18 most work on hydrophobic interactions, phenyl/aromatic on pi-pi bonding etc.

If we look at C18 for Reverse phase we follow the rule that:
Stationary phase is non-polar
Mobile phase is polar
Therefore your elution order is; most polar analytes elute first (flushed off with mobile phase) and non-polar are retained longer on the column. For a C18 column you can look at LogP values which give you an idea about polarity of a compound (this is based on the preferentially solubility of the compound into either water or octane - immiscible solvents, hydrophillic would be solubility in water, hydrophobic would be solubility in octane).
A lower Log P value <0 indicates a hydrophillic (polar) compound, a Log P >0 indicates a hydrophobic compound.

RP-HPLC is mostly used for hydrophobic (non-polar) due to elution.

Looking at your analytes they have the following LogP values:

acetophenone (1.58), nitrobenzene (1.85), benzene (2.13) and toluene (2.73). The elution order is acetophenone - nitrobenzene - benzene - toluene. These LogP values can be found on ChemSpider - great place for information! Its getting a little too chemistry based for me, but it may be that you have to take into the account the conjugation of the benzene ring Vs the -R chains. The acetophenone only has one electronegative group (C=0), whereas the nitrobenzene has two oxygens).

Common RP solvents include; Water > Methanol > Acetonitrile > THF
Going from most (weakest) polar > least polar (strongest).
The more aqueous content you have on a RP-HPLC C18 column the more retained something non-polar will be, if you ramp up the organic solvent this will cause your analytes to elute quicker. Think like dissolves like (or like-attracts-like).

So if you switch between solvents your retention times shift. Strength is polarity based, stronger solvents been less polar. This is reflected by the LogP values
Methanol (-0.77) > Acetonitrile (-0.34). ACN is closer to 0 and therefore less polar. But, care needs to be taken between solvent strength when changing solvents, solubility of samples, miscibility of solvents and also if a method is isocratic or gradient.

Remember HPLC is for non-volatiles, GC for volatiles (as a general rule).

Hope this helps!

[chromatographer1]

Wax phases, 50% phenyl methyl silicone, 5% phenyl methyl silicone, OV-1301 and OV-1701 are all good phases. Even a simple 100% methyl silcone (nasty peak shape for the nitrobenzene though) would be useful.

If you really need to know the elution order, look up the McReynolds constants for each phase in your library. The number assigned to each analyte is based on an alkane sequence.

best wishes,

Rod