First, you need to swell the LH-20 before packing the column in the solvent you will use. It can swell a large amount. The solvent really depends on what solvent elutes your active compound, I like to use methanol.

This link lists solvents, and how much they swell, and other information as well: https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314735988470/litdoc56119097AD_20110830235244.pdf

The sample will be dissolved in a solvent compatible with your mobile phase (ideally in your mobile phase) and added by pipette. 

Do not try to run the column too fast. LH-20 compresses easily if the flow gets too high, causing high back pressure, which causes more compression.. If using a glass column, just let gravity let the solvent through- do not speed up the flow rate with compressed air (like is done for flash chromatography).

If you have an automated flash chromatography system, you can attach the column if the system can run at a slow flow rate (1 through 5 mL/min, 5 mL/min for a large LH-20 column). This lets you use the detector and fraction collector in the flash system as was done here: http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN23_Self-pack_Column_Options_for_the_CombiFlash_Rf.pdf

Once packed, don't let the column dry out; it will form "cracks" from shrinking, and those cracks will ruin your chromatography.

As for regeneration, I've heard of people washing the LH-20 with a solvent compatible with water (unpack the column first), then water containing a little bleach, then water again. They let the LH-20 dry out. I haven't done this myself.

Okay, Thank you Jack Silver. I mean by regeneration that whether after running the column once , can i re use the same column for another loading again. Will there be any contamination or poor separation as compare to the last one. For example if i have large sample size and column filling is small because of small amount of stationary phase. Can i load my sample more than one time on the same column ?

Samina, you can _probably_ reuse the column for the same sample, but it depends on the nature of the late eluting compounds. Some stuff is very well retained and won't elute later, some compounds will. I'm sorry for the vague answer, but it really depends on the mixture. That is one reason I liked to be able to run the mixture on a system with a detector- I can see if there is anything still eluting.

Here is the data sheet that tells you all you need to know about Sephades LH20:-

https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314735988470/litdoc56119097AD_20110830235244.pdf

I will be using Sephadex G-25 for a protein separation practical in just over a week's time. We routinely regenerate Sephadex G-25 by reverse flushing it out of the 10ml column we use, through a coarse seive to remove any filters, into a beaker. We pass this through filter paper, then add 0.4M NaOH(aq) and stir. This is then filtered to dryness (the 0.4M NaOH(aq) can be re-used) then washed thoroughly with deionised water and filtered again before storage in 20-100% Ethanol. Works as new!

The regeneration protocols can be found on the GE Healthcare website, for each type of sephadex.

Okay . Nice information. Thank you sir.

Which ratio Sephadex LH- Sample is better to use to not overload the column?

20:1, 50:1?

@ Nuria- good question. The stuff swells easily, I'd guess, by weight, it is around 10:1 though 20:1 dry weight.

In the link to Sephadex LH-20 on the GE Life Sciences website (see my earlier response) there is a table which suggests that each gram of Sephadex LH-20 will swell to 4.0-4.4mls volume in water, which agrees with our experience using Sephadex G-25 (2.5g dry weight≡10mls volume after swelling fully. It will ultimately depend on the solvent that you are using - see their table for Sephadex LH-20.

Thank you @Jack Silver and @Zoë Gaffen. I applied the sample at 35:1, solved in the same concentration solvent as the column was packed.

The column is flowing so so slowly (0,5cm/h). Do you have a tipp how to increase the flow? sould I change the solvents ( now is 8MeOH:2H2O) and I would like to run isocratic.

The sample is water soluble.

thank you in advance

Check whether you have an air bubble impeding the flow (these can lodge in the luer tip of the column or in the hub of any attached tap) - if so, attach a tubing between a syringe and the column outlet, and withdraw less than 1ml of liquid, ensuring that a head of liquid is still covering the top of the column packing. This should immediately increase the flow dramatically, if that was the cause of your problem.

Aside from what Zoe said, those columns tend to run slow. How long is the column?

One could use just a little air pressure or pump, but very, very, carefully. These columns compress easily. If one tries to run them too fast with a pump or air pressure, they get compressed, the back pressure increases, and they compress some more in a cycle. Please note in my response earlier in the thread that I ran them at a very slow flow rate to prevent the compression. If the column compresses too much, it won't run at all, and one will need to start over again.

The column is 82cm long and 3,5cm diameter. It's runing at 0,5cm/h, that means 0,1ml/min.  It was used sephadex, and it seems that ich compress more than it should. There are not bubbles, so I should wait....

Thanks so much for your answers Jack silver and Zoë Gaffen

I have found another link, supplied by the manufacturers of Sephadex LH-20,

which goes into detail about how to calculate optimum flow rate (yes, it is rather slow for this packing material) and recommended column re-equilibration and re-generation between separations:-

https://cdn.gelifesciences.com/dmm3bwsv3/AssetStream.aspx?mediaformatid=10061&destinationid=10016&assetid=11273

I hope it helps.

Hi I am palnning to use Sephadex column for de-colorization of ionic liquid. Do you think it will work? From where I can buy cheap sepadax column?

Sephedax HL-20 has become a block not powder in the purchase box How do I treat it?

@ Norah-

It seems that it may have been mixed with a little solvent such as water, and dried. Break it into chucks, then make a slurry in the solvent you need to use, and run it as usual.

Is it possible that your LH-20 may have been used, and someone placed it in the original container? It should still run properly.

This Sephadex LH20 should be in a small bead form, white with faintly rose color. Regeneration may be done by 4% sodium bicarbonate aq. solution.

I agree that it is indicative that the solvent that it was originally suspended in has evaporated, if the Sephadex has formed a solid lump or 'block'. It can be easily resuspended in 20% Ethanol(aq) or similarly bacteriostatic solvent, for storage at 4-8°C, prior to column packing, followed by equilibration in the chosen solvent (column bed volume will be slightly greater in an aqueous buffer).

PD-10 columns can be bought inexpensively, and re-used, for a simple low-pressure, 15mm id, 10ml Sephadex Column. The luer tip facilitates addition of a tap, to start/stop the flow.

SephadexLH-20.pdf

I use 750 ml of wine to obtain condensed tannin fractions by gradient of methanol and acetone, I also use compressed air to increase flow rate. The column compresses, yes, but I'm not entirely interested in better seperations. Tannins bind strongly to Sephadex anyway, but you can use MCP or TLC to monitor fractions as they collect. I'll use either ethanol or isopropanol to wash the column from any red anthocyanin residues, because anthocyanins are soluble in water and alchols, but the inherent structure of the hydropropylated dextrans leads me to believe that propanol will do the job; LIKE DISSOLVES LIKE.

Chemistry is so wonderful...

As long as your column doesn't crack!

Check its specification and material data sheet. Don't use water at least;

Recently we came up with a sticky challenge; where by mistake my students dissolved LH-20 Sephadex in water for column preparation/packing buy not knowing that this will get dissolved. As Sephadex is an expensive material, we planned to dry it in Freeze drier, but after drying when we take it out from drier, it absorbs moisture from air and get sticky. We tried with rotary as well but get the same effect. After successive failures, we thought to dry it solvent - solvent extraction methodology. In this experiment, we dissolved LH-20 in distilled water, added equal volume of n-butanol in it and started its drying through rotary evaporator. As soon there is less than half of solvent left, we added equal amount of methanol it and dried it completely through rotary evaporator. Luckily, we get it dried with no stickiness and got a smooth powdered material. Samina Aslam

Muhammad Jahangir - Are you sure that was Sephadex LH-20 that your student used? LH-20 doesn't dissolve in water, and it isn't sticky from absorbing water. someone posted the instructions as a PDF a couple of replies above. Those instructions specifically mention how much LH-20 can be expected to swell in water.

Thank you for the wealth of information shared.

I want to run a sephadex column for the separation of polyphenolic cpds (hydrolysable tannins) and have set up a small column to study the process. Your answers have been handy and helpful.

I will like to know if it is okay to dissolve my extract (aqueous) in 50% Ethanol even though i want to run in 100% Ethanol, followed by 10% Methanol, 30% Methanol .....100% Methanol.

Thank you

Jonathan Jato , if you are asking whether sephadex is OK in contact with 100% Methanol or Ethanol, the answer is yes. It won't be harmed by those solvents - I routinely use 100% Ethanol for long-term storage of the regenerated sephadex that I use for my undergraduate Protein Separation practical classes. However, I can't confirm whether your solvent progressions will work well for your intended separation(s) or not.

Jonathan Jato , as Zoë Gaffen said, the resin is compatible with the solvents. However, LH-20 swells up when exposed to solvents. The amount of swelling is different for different solvents. There is a PDF file posted earlier in the thread, and it tells how much LH-20 swells in different solvents. Referring to those instructions, you'll find that methanol causes it to swell more than ethanol, so an ethanol to methanol gradient is bad. The LH-20 will swell more as the methanol concentration increases, causing higher back pressure, and reduced flow rates. This is why LH-20 is generally run isocratically.

Thank you Zoë Gaffen and Jack Silver