Chromatography Forum

it is odd that people do not recommend use of mixture solvent,only use one pure solvent. how can they gurantee complete elute of analytes in sample?

You can *never* guarantee complete elution in liquid chromatography.

small molecules sample after SPE in C18 can be completely eluted out with up to 100% gradient organic solvent.

You can *never* guarantee complete elution in liquid chromatography.

Gradient should do better job in complete elution of sample than isocratic within reasonable time. right.
so how can i improve this on LH-20, since gradient elution will constantly change bed volume LH-20.

You can *never* guarantee complete elution in liquid chromatography.

small molecules sample after SPE in C18 can be completely eluted out with up to 100% gradient organic solvent.

Usually, but not always. That's why you will see "regeneration" procedures in the literature that recommend flushing reversed-phase columns with solvents such as methylene chloride.

That said, Sephadex LH-20 is a different animal. It was never designed for use under HPLC conditions, and lacks the rigidity necessary to tolerate large changes in mobile phase composition (not to mention pressure!). That means that many of the procedures we take for granted with HPLC columns (gradients, high flow rates, backflushing the column, large changes in solvent types (so long as they are miscible!) will not apply.

If you have strongly bound material in your typical mobile phase, the best approach may be to unpack the column, clean the packing "batchwise" with an appropriate solvent, and then repack the column. What constitutes and "appropriate" solvent, of course, depends on what kinds of strongly-bound contaminants you expect.

did you see any good review paper on LH-20, i did not see any.
could you recommend one of you know any.

small molecules sample after SPE in C18 can be completely eluted out with up to 100% gradient organic solvent.

Usually, but not always. That's why you will see "regeneration" procedures in the literature that recommend flushing reversed-phase columns with solvents such as methylene chloride.

That said, Sephadex LH-20 is a different animal. It was never designed for use under HPLC conditions, and lacks the rigidity necessary to tolerate large changes in mobile phase composition (not to mention pressure!). That means that many of the procedures we take for granted with HPLC columns (gradients, high flow rates, backflushing the column, large changes in solvent types (so long as they are miscible!) will not apply.

If you have strongly bound material in your typical mobile phase, the best approach may be to unpack the column, clean the packing "batchwise" with an appropriate solvent, and then repack the column. What constitutes and "appropriate" solvent, of course, depends on what kinds of strongly-bound contaminants you expect.

did you see any good review paper on LH-20, i did not see any.

I'm not sure exactly what you're looking for. This is a product that has been around for 40 years. A Google search for "Sephadex LH-20" turned up over 200,000 hits, including this one:
http://www.skkupharm.ac.kr/krlee/erp/er ... exLH20.pdf

There has to be *something* useful in there!

i just do not know why there is partition mode in LH-20 chromtogram.

did you see any good review paper on LH-20, i did not see any.

I'm not sure exactly what you're looking for. This is a product that has been around for 40 years. A Google search for "Sephadex LH-20" turned up over 200,000 hits, including this one:
http://www.skkupharm.ac.kr/krlee/erp/er ... exLH20.pdf

There has to be *something* useful in there!

i just do not know why there is partition mode in LH-20 chromtogram.

Why would there *not* be partition (or adsorption)? Minimizing "secondary" interactions with the stationary phase is probably the hardest part of doing GFC of proteins.

I thought stationary phase in LH-20 is solid, so it can not be called as partition chromtography.

i just do not know why there is partition mode in LH-20 chromtogram.

Why would there *not* be partition (or adsorption)? Minimizing "secondary" interactions with the stationary phase is probably the hardest part of doing GFC of proteins.

"partition" and "adsorption" are simplified labels that we apply to complex processes. Each describes a part of the whole. The contents of the following link may help put things in perspective.

http://www.wordinfo.info/words/index/in ... =B&spage=3

jiang295, you can look into attempts to explain HILIC to find out why there might be partition with "solid" stationary phases.

thanks.

jiang295, you can look into attempts to explain HILIC to find out why there might be partition with "solid" stationary phases.

so actually, it is the same as C18 partition mode.
treat stagnant mobile phase as stationary phase.

jiang295, you can look into attempts to explain HILIC to find out why there might be partition with "solid" stationary phases.

do you guys think LH-20 stationary phase is similar to cyno column?

You can *never* guarantee complete elution in liquid chromatography.