Chromatography Forum

Dear Members,

Fact # 1:
Weak solvent strength in RP-LC means more polar solvent.

Fact # 2:
Chromatographic separation is based on affinity to stationary phase and solubility in mobile phase.

Fact # 3:
When we develop RP-LC method for a component, polar substance could not be enough retained to the column packing. To get sufficient retention, sometime we should reduce the solvent strength.
Logically, if we use more polar mobile phase then the polar substance more difficult to retain in the column due to it is more soluble in the mobile phase. Did I miss something? Is there any other mechanism in the separation?

Best regards,
Siswanto Tanuatmojo

I would rephrase your "Fact 2" to say that separation is obtained as a result of the differences in partition coefficients among your analytes given the combination of mobile and stationary phases you've put them in. (If you have solubility issues, you're not separating things chromatographically.)

The "other mechanism" also pertains to the partition coefficient but it would help to think in terms of hydrophobicity which is a little more encompassing than polarity. Aside from lowering % organic, you can also switch from ACN to MeOH or switch stationary phases to drive your unretained analytes into the stationary phase more.

Logically, if we use more polar mobile phase then the polar substance more difficult to retain in the column due to it is more soluble in the mobile phase.

For RP, it may not true if you change 90% Methanol aqueous to 70%, where the latter is more "polar" but the former may be better for dissolving organics.
Let me know if I am wrong, Thanks

If your sample isn't in solution, you aren't doing chromatography. Whatever you choose as your mobile phase system, your sample has to be solubile in it at whatever concentration you intend to inject. For gradients, this may well cover a considerable range of organic concentrations.

I would rephrase your "Fact 2" to say that separation is obtained as a result of the differences in partition coefficients among your analytes given the combination of mobile and stationary phases you've put them in. (If you have solubility issues, you're not separating things chromatographically.)

The "other mechanism" also pertains to the partition coefficient but it would help to think in terms of hydrophobicity which is a little more encompassing than polarity. Aside from lowering % organic, you can also switch from ACN to MeOH or switch stationary phases to drive your unretained analytes into the stationary phase more.

Between ACN and MeOH. MeOH being more viscous than ACN will longer the retention time. But looking in to Snyder values of polarity for two, ACN 5.8 Methanol 5.1. methanol being less polar than ACN should elute the compound faster. Am i wrong somehere?

Or the values ACN 6.20 methanol 6.60 for polarity removes the contradiction of above statement.

Please clarify!

Thanking you

Regards

Amaryl

I would rephrase your "Fact 2" to say that separation is obtained as a result of the differences in partition coefficients among your analytes given the combination of mobile and stationary phases you've put them in. (If you have solubility issues, you're not separating things chromatographically.)

The "other mechanism" also pertains to the partition coefficient but it would help to think in terms of hydrophobicity which is a little more encompassing than polarity. Aside from lowering % organic, you can also switch from ACN to MeOH or switch stationary phases to drive your unretained analytes into the stationary phase more.

Considering your analyte nonpolar...
Between ACN and MeOH. MeOH being more viscous than ACN will longer the retention time. But looking in to Snyder values of polarity for two, ACN 5.8 Methanol 5.1. methanol being less polar than ACN should elute the compound faster. Am i wrong somehere?

Or the values ACN 6.20 methanol 6.60 for polarity removes the contradiction of above statement.

Please clarify!

Thanking you

Regards

Amaryl

Dear Members,

Fact # 1:
Weak solvent strength in RP-LC means more polar solvent.

Fact # 2:
Chromatographic separation is based on affinity to stationary phase and solubility in mobile phase.

Fact # 3:
When we develop RP-LC method for a component, polar substance could not be enough retained to the column packing. To get sufficient retention, sometime we should reduce the solvent strength.
Logically, if we use more polar mobile phase then the polar substance more difficult to retain in the column due to it is more soluble in the mobile phase. Did I miss something? Is there any other mechanism in the separation?

Best regards,
Siswanto Tanuatmojo

we have to consider the polarity of mobile phase. Water being more polar than ACN and MeOH...if we increase the content of organic in mobile phase the mobile phase polarity is decreasing and ideally the polar compound should have more interaction with C18 to provide you with reasonable retention.

is it fine? Hope it is:)!

For polar compounds ion chromatography can be a better alternative.

Regards

Amaryl

amaryl, if you increase the organic you displace the polar substance even more from ther stationary phase (lower retention, not higher; chances are very high that you will be far away from precipitating your analyte with the org. modifyer at the concentrations involved). Take another look at DR´s statement, that´s all you need to know.

Also - viscosity has little to do with retention or k for a given analyte. All viscosity does (from a practical point of view) is limit how fast you can run your samples through your system.

Syx:
The polariy of a analyte is somewhere between the stationary and the mobile phase. In RP chromatography the mobile phase is much more polar than the analyte. If you have "polar" analytes, you have to increase the polarity of the solvent in a way that analytes polarity is still half way between stationary and mobile phase.
Or the thoer way around:if "polar" analytes (less hydrophobic would be a better term) are not retained enough on the column, you have to use a weaker solvent.
Amaryl:
Viscosity has certainly some influences on RP-HPLC but it is not the reason why elution with methanol takes longer. Take it as granted that Acetonitril in most cases is a stronger solvent.

Syx:
The polariy of a analyte is somewhere between the stationary and the mobile phase. In RP chromatography the mobile phase is much more polar than the analyte. If you have "polar" analytes, you have to increase the polarity of the solvent in a way that analytes polarity is still half way between stationary and mobile phase.
Or the thoer way around:if "polar" analytes (less hydrophobic would be a better term) are not retained enough on the column, you have to use a weaker solvent.
Amaryl:
Viscosity has certainly some influences on RP-HPLC but it is not the reason why elution with methanol takes longer. Take it as granted that Acetonitril in most cases is a stronger solvent.

Well I agree with you sir. ACN being a strong solvent elutes analyte faster than MeOH.

As mentioned by Siswanto, polarity and solvent strength have reverse relation. So can we conclude that MeOH is more polar than ACN i.e the values 6.60 and 6.20 holds.

And the Synder values for polarity (ACN 5.8 Methanol 5.1) does not holds if we consider solvent strength ( viscosity as i tried) to account the faster elution with ACN.

Thanks

Regards

Amaryl

amaryl- I dont feel viscosity has something significant to do with HPLC separation. Further, the mobile phase pumping velocity is user controllable parameter (just higher back pressure for more viscous fluid).

Simple polarity may not define the correlation between retention time and polarity of mobile phase. Rather various thermodynamic properties ( hence molecular interaction) will define retention time. If polarity is only property defining RT, probably we dont need wide array of mobile phase solvents simply because two solvent mixture (in appropriate ratio) can give some desired polarity.