Normal Phase HPLC - purification of non polar Nat prod.

[dan42]

Hi all

Im so desperate and grrrrrrrrrrrrr.
I am currently running normal phase HPLC (Hexane and Ethylacetate) to separate non polar constituents. I need to find a good method any suggestions???using these solvents? When I use 5% Ethyl acetate everything in unretained grrrrrrrrrrrr. When I use 100% hexane, nothing come off it all sticks. My samples are made up in 100% ethyl acetate
Ive tried today switching to Isopropanol and Hexane. However changing Line A to Isopropanol was disasterous it seems that it is too viscous, pressure exceeds 3000 psi????? Why?
What are some good solvent systems to separate non polar compounds (unknowns) isocratic or gradients?
Please helpppppppppp

Cheers

[Alex Buske]

First of all: dissolving sample in 100% ethyl acetate is not a good idea. try your solvent mixture.
Ethyl acetate has quite a strong UV absorption, so IPA would be a better choice.
3000 psi is not a problem. Any pump I know is specified >5000 psi.
Next point: If you run gradients or work with Soovent mixtures like 95/5 or 98/2, don't use pure solvents on the Pumps. Prepare mixtures like 90/10. No pump delivers mixtures <5% accurately, especially in normal phase.

Alex.

[tom jupille]

Or use a lower % ethyl acetate. Retention behavior in normal-phase chromatography (retention vs % strong solvent) is much closer to log-log than to log-linear (the situation in reversed phase). The implications of this:
- in reversed-phase, the retention time changes by about the same factor for every 10% change in %B.
- in normal-phase, the retention time changes by about the same factor when you change from 0.1 to 1% as from 1% to 10% as from 10% to 100%.

Bottom line: most of the "action" occurs within the first 10%, so it's not unusuala to see NP systems with only a few% strong solvent.

[dan42]

First of all: dissolving sample in 100% ethyl acetate is not a good idea. try your solvent mixture.
Ethyl acetate has quite a strong UV absorption, so IPA would be a better choice.
3000 psi is not a problem. Any pump I know is specified >5000 psi.
Next point: If you run gradients or work with Soovent mixtures like 95/5 or 98/2, don't use pure solvents on the Pumps. Prepare mixtures like 90/10. No pump delivers mixtures <5% accurately, especially in normal phase.

Alex.

Thanks, heaps for your suggestions.

[HW Mueller]

What is the reason for using normal phase?

[dan42]

Well thanks guys, just one more query..

It seems that Ethyl acetate might be to much of a strong solvent to use with hexane. Would using dichloromethane with Ethyl acetate seems a better choice of solvents to use in terms of compounds sticking on the column longer?

Cheers

Will intend to use this tomorrow!!!

Thanks again for both your input

[tom jupille]

Probably not. Since MeCl2 is more polar than hexane, it would be a stronger NP solvent from the start. MeCl2 in hexane might be a better bet.

[dan42]

What is the reason for using normal phase?

The reason is I have non polar natural products which only dissolve in non polar solvents. When I tried to use reverse phase HPLC (ACN + Water) These compounds (which were only sparingly soluble) eluted toward the end of my run. Trying to get good separation was difficult.
Next problem was using Ethyl acetate and Hexane (Normal phase). THese compounds which were dissolved in Ethyl acetate all eluted at the start of my run (35% Ethyl acetate/hexane). When going to 5 % or less, I achieved better separation over 15 minutes but nor base line resolved separation. I had to go down to 0.3% Ethyl acetate to get these compounds to come off the column. THey wouldnt come off with 100% Ethylacetate.

What I need is a good solvent system that seprates non polar compounds well. ANy ideas????

[JM]

For preparative HPLC , it is no good to seperate all compunds of extrem polarities in one run. I would suggest to 1st segregate crude fractions of non-polar , mediumn polar and polar using simple silica flash column using wash cycle of 3 polarity solvent system. Sample loading may not be a problem as you can adsorbe your mix on silica with whatever solvent, dry and fill on the top of your column.

ones you segregate different polarity fractions of your mix , you can resolve them using prep HPLC with matching polarity mobile-phase.

am I clear on this?? Good luck!!


JM

[Alex Buske]

As you are not restricted to RP and don't care much for the UV, you have all the possibilities of NP separation. Try to find a paper from Snyder around 1974 "Properties of commem Liqiuds as solvents" or so and a later paper by someone else on "prisma" TLC method development.

[HW Mueller]

dan42, you mean 100% hexane instead of ehyl acetate up there?
Just wonder what your compounds would do with ethyl acetate as mobile phase on a RP column.