By ieatapple on Monday, June 14, 2004 - 07:16 pm:

I have one question.


reverse-phase HPLC conditions in one paper I read are as follows:
C18 column
The eluents were 100% ethyl acetate(solvent A), and 90% acetonitrile(v/v) in water( sovent B).
Flow rate was 1 ml/min with a gradient elution system of 0-1 min, 0%B; 1-30 min, to 60% B; 30-51min , 60% B; 51-55 min, to 0%


My quesion is

In reverse phase chromatography, since the stationary phase is nonpolar,nonpolar eluent is stronger, isn't it?

100% ethyl acetate (solvent A)
90% acetonitrile(v/v) in water( sovent B)
Which is more polar? I think B is more polar, isn't it?

But in the paper, the author use the gradient elution that increase the percentage of B during the analysis. I think we should increase the percentage of stronger solvent(A) in gradient elution.

I am really confused about it. Do you think in reverse-phase gradient elution, we can decrease the strength of eluent? Isn't it so unreasonable?

Thank you for your help!

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By Anonymous on Tuesday, June 15, 2004 - 01:42 pm:

acetonitrile is non-polar and usually you increase acetonitrile. so I think you are ok.

however, I have seen a method which starts with 100% acetonitrile and after a while small amount of water was added. Water slowed down everything and final separation took place during the slowing.

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By Chris Pohl on Tuesday, June 15, 2004 - 04:03 pm:

ieatapple,

I think you are correct. While there is no generally agreed upon parameter that can be used to unambiguously answer this question, all of the tabulated values point in the opposite direction. For example, the dielectric constant of ethyl acetate is 6.02 and the dielectric constant of acetonitrile is 37.5. The dipole moment for ethyl acetate is 1.88 whereas the dipole moment for acetonitrile is 3.44. Finally, the polarity index value for ethyl acetate is 4.4 while the polarity index value for acetonitrile is 5.8. Taken together, these three parameters all show acetonitrile to be more polar than ethyl acetate suggesting it should be a less potent solvent than ethyl acetate from a reversed phase point of view. The only parameter which suggests the opposite is the elutropic value for the two solvents on alumina. In this case, the value for ethyl acetate is 0.58 while the value for acetonitrile is 0.65. This suggests that the separation you are performing is actually normal phase via interaction with residual silanols.

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By Anonymous on Sunday, June 20, 2004 - 07:11 pm:

Some strange things can happen with polymer samples, but otherwise I agree with Chris.