That should work. If the compound is soluble in 10% water in methanol, it may elute earlier. Dry loading will give the best resolution in my experience. You'll want to wash with 5 to 7 column volumes of your starting solvent (10% methanol in water) to equilibrate the column; C18 seems to take longer than silica in this respect.

Good on starting with 10% methanol- this will prevent "phase collapse" where the C18 doesn't interact with the mobile phase anymore because it is too polar.

I've written an app note describing loading techniques for pre-packed C18 flash columns:

http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN88_C18_Flash_Column_Loading.pdf

Thanks alot Dr. Jack I'll do so and I hope it will work as I have 5 Polar Flavonoides in my Fraction very difficult in their separation

It seems to be another Question ? When can I change the mobile phase from for example10% methanol to 20% methanol and so ,as i won't be able to Check the fractions by TLC until get concentrated by The Lyophilizer

I'd run at least one column volume, two or three would be better between steps.

To determine the column volume, pask a small column with a known weight or volume of packing (I tend to use weight if possible); pack it wet with methanol. Let the column drain to the top of the packing., then get a fresh flask and place it under the column. Drain the column into the flask until it runs dry, then measure the volume of methanol in the flask. This volume is one column volume. you only need to do this once since it scales. A small column of ~20g will work well. you can re-use the media for your purification column.

Since you know the amount of media used in your "test" column, and you know the amount of media used when you packed your purification column, you can use the ratio of the columns determine the column volume. If you scale up, you can just scale up the step sizes proportionally and your compound will still come out at about the same time. If you make a column with twice the amount of C18, your steps should be twice the volume of solvent and your compound will elute in pretty close to the same number of column volumes.

Thanks Dr. Jack This is a good idea I'll try but from your experience what was approximately the column volume of a small column of 20 g ? just to have an idea

One another question, Can I monitor the fractions of C18 silica on normal silica TLC or not?

Well- a 26 gram C18 column, dry-packed by machine, has a column volume of 26.4 mL. I measured this by weighing the column packed, and again after filling with a solvent; I then measured the density of the solvent. Please note that Teledyne ISCO manufactures flash systems and pre-packed flash columns, including various types of C18 columns. That's how I could pull up this number fairly quickly. Your value could be very different from mine.

You can probably monitor the fractions by silica TLC but you'd need to work out a solvent system so the desired compound is resolved from the impurity. Lots of compounds can be purified by either method.

I read that your compound is fairly polar (soluble in 10% methanol in water) suggesting a dichloromethane/methanol solvent system may work. Hexane/ethyl acetate with a large percentage of ethyl acetate might also work. This is only a guess based on what I read.

Ok Thanks Dr. Jack ,Really the discussion with you was very fruitful

Unfortunately, my compounds resolve well with only BAW solvent system (4:1:5)

Dear Dr. Jack silver,

Nice discussion i have gone through your topic.

Have you protocol to prepare agarose beads column for metal affinity chromatography of His-tagged recombinant proteins?

with regards

Dear Dr. Rashid;

Thanks for your kind words, but I haven't worked with that material. If it's like Sepharose (I used Sephadex LH-20), it needs to be swollen before use, but this is a dextran gel, not agarose.

Sorry, I wish I could provide meaningful assistance!

Dear Dr Jack Silver,

thanks for your information. Yes the column is prepared with sepharose. Have you any idea how to use sepharose properly and for further utilization for different purposes.

Please provide me the literature for the said purpose.

with kind regards

Dr Rashid, you may start here:

http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences/brands/sepharose/

If it isn't preswollen, you need to place it into the solvent you will use, then let it swell for a few hours, then pack your column. Note that a little goes a long way- it swells up like popcorn. The first time I used it I wound up with enough swollen Sephadex for about 5 or 6 columns!

Dear Dr. Silver I used Sephadex LH-20 (mobile phase was Methanol )many times but I didn't succeed to separate pure componds from it especially flavonoides so How can I know that Sephadex LH-20 will be suitable method for separation of compounds

Thanks Dr. Silver for your kind information

Dear Dr. Malash;

I never got pure compounds from LH-20, but it does a good job at "simplifying" a mixture. The attached poster from last years' American Society of Pharmacognosy meeting gives a good example:

http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Purification_of_Phenolic_Flavonoids.pdf

The center column of the poster shows what the mixture looked like on a couple of different types of C18 before Sephadex; the misture was run on LH-20, and fraction #4 was run on the C18 again. The chromatogram is much simpler, and allowed me to obtain the pure flavanoids on the C18Aq column.

To give a succinct answer to your question, I ran chromatography (TLC is also useful, depending on what your are purifying), determined the mixture was complex, then ran LH-20 to make it easier for the next purification step.

Thanks Dr. Silver , ok If I have TLC sheets of reversed phase C18 silica can I use AlCl3 as a spray reagant and NH3 vapours and Anisaldehyde/Sulphoric as we use with normal phase silica?

Dear Dr. Malash;

Do not use sulfuric acid with C18 TLC plates since the C18 will char and you won't see anything- it will all be black.

I think you can use the AlCl3/ammonia vapors since is shouldn't react with the plate. Best to just try it with a plate with nothing run- just spray it and try it. You'll know very quickly if the plate will react.

Thanks a lot Dr. Silver for your valued answers

One last question :Do I put a piece of cotton under my sample and one another above it in the column of C18 silica?

Under you sample or your column? I assume you are dry-loading your sample (absorbing on C18 or Celite and placing the dry material on top of the packed column)? I tended to use glass wool under the column to keep sea sand in place, then packed the column on top of the sand. I would load the sample, and put more sand on top to protect the bed during solvent changes.

yes I will make dry loading but I meant between the sample and C18 bed?

No- it would create a void where compounds can be dissolved, It would tend to cause tailing. It would act like a void in the column.

Oooh Thanks for your explanation coz in our department they tend to put a cotton under the sample but I didn't know why .

Nice for asking you even the finest steps

Thanks again

Dr. Malash- I'm guessing they are trying to reuse the C18 or other packing; anything that retains will tend to stick to the media the sample is adsorbed upon. They may be "washing out" the "contaminated" media before emptying the main column.

Thanks Dr. Silver for your explanation

ok let me be sure about something :C18 Silica is reusable after I make washes for it 5-7 times with the first mobile phase as you said before but the amount of C18 silica the sample is adsorbed on is discarded because it will be contaminated even after several washes?

Is this right?

Dr. Malash; that C18 might be contaminated, it is usually discolored from material that either isn't soluble in your solvent system or managed to get to the underlying silica and get stuck there. You might be able to remove some retained material with dichloromethane followed by methanol (dichloromethane is immiscible with water and the methanol wash lets you use aqueous solvents immediately). This allows you wash very non-polar compounds off. Anything stuck to the silica isn't likely to bother you, especially if it is used for the same mixture next time.

Thank you very much Dr. Silver .

Really your answers were so precious for me and all of researchers who will read this topic.

Now I finished my experiment on C18 column, I reached 100% methanol and washed with dichloromethane but the C18 is still yellow from my sample, How can I make the C18 return to its original offwhite color?

You might not be able to clean it completely- remember something may be stuck to the underlying silica. You could try a hexane wash, which is about as non-polar as you can get. It it is still yellow after that, it almost certainly won't elute into you other samples.

Thanks Dr. Silver but this will not affect the adsorption capacity of the C18 silica for the next sample?

No- it is chemically bonded and the only thing that would come off is any C18 that was already going to come off because it was hydrolyzed. Remember that you'll need to go back through DCM and methanol to make the column useful for aqueous C18 again. Save & analyze your hexane wash in case you got something from your plant extract that might be interesting.

Honestly, the DCM should have gotten the non-polar compounds off, I'm pretty sure the yellow is stuck on the silica.

yes this explains why I found some C18 silica in the first wash with 10% methanol in water before I add my sample this made me suspect the size of the cotton under the column bed that may made some leakage to happen but as you said this C18 silica come off because

it was hydrolysed.

But now after I was able to resolve my compounds by mobile phase BAW on TLC ,it's now only suitable for the first fractions that came off the column while the later fractions don't resolve with any of these mobile phases:

1- CHCl3:MeOH 8:2

2- hexane :EtOAC 2.5:7.5

3- Toluene: EtOAC 1:9

4- EtOAC : MeOH: H2O : Formic acid 7.5:1.5:1.25:one drop

Remember that I have polar compounds so which mobile phase also can I try?

Mixture of polar & non-polar? I do the water/methanol gradient and go into methanol/DCM gradient if needed. you may need to use another column to further resolve the collected fractions, with another media sich as silica or alumina or LH-20

yes mixture of polar and non polar ,I found a major compound (yellowish green after spraying with AlCl3) in a group of fractions so if I want to make a minicolumn with silica, which mobile phase can I use as chloroform : MeOH don't give a good resoltion of my compounds?

Hard to say- you should run TLC with various solvents solvents. If it works on TLC, it will sork on a silica column. Keep the Rf less than 0.5 or the compounds will elute too fast from the column. Try acetone or ethyl acetate with methanol, or try HILIC- acetone/water or acetonitrile/water. HILIC on silica works well with good quality silica (don't use silica G). Also, don't use basic solutions- keep the pH less than 7.5 or the silica will dissolve.

I suggest this because these solvents are in different selectivity groups than DCM/methanol.

Details of solvent selectivity can be found in this free book:

http://www.isco.com/flashguide/

Example of silica HILIC (Figure 5):

http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Strategies_for_the_Flash_Purification_of_Highly_Polar_Compounds.pdf

Please note I work for this company.

If one compound eluted from C18 Silica Column, Does it need to be purified on another column such as Sephadex LH-20 ,or can be analyzed directly?

Depends on how clean it is. If the compound went through a purification step or two prior to C18, it is likely clean.

Thanks Dr. Jack

I have 2 spots eluted from the C18 column in a group of fractions and dissolved them in 90% methanol in water and was filtered before adding on a column of Sephadex LH-20 then elution was made by 100% methanol , one compound was separated but the other remained as a band on the top of the column , I increased the polarity with water (As i know that this compound is soluble in water) till i reached 80% methanol but no avail ,it didn't even moved from its place ,Should I increase the polarity to 50% methanol in water or remove this band and soak it in water then filtering off the sephadex?

I'd remove the band and soak it because Sephadex swells differently in different solvents. I forget if it would swell more with increased water-if it swells, you may get more back pressure & slower flow and maybe even break the column. Maybe post this question as a new topic so others that use Sephadex more than I and have better knowledge can answer?

Thank you very much Dr. Jack ,I prefer this solution , and I'll ask in a new topic about changing the mobile phase in columns of sephadex

I put sand on top of my C18 flash the first time I used/made it to keep the bed stable.  I want to reuse it for same mixture - can I dry load sample on top of sand or do I need to figure a way of getting the sand out? 

How much sand are we talking about? Also, what substrate are you using for dry-load? It will broaden the band, but if only a little, it could cause problems. If you want to unpack the column later to wash and re-use the C18, that bit of sand will be hard to remove if there is something (dry-load) on top of it.

About. 1 cm of sand. I use C18 to dry pack the fraction. I don't plan to reuse the C18 for anything but this same mixture - but I will end up having to reuse this column for the same mixture likely 2-3 more times.. 

Depending on the width of you column, maybe put a bunch of glass wool or cotton ontop of the dry load? This could protect the bed and you can "fish" it out between runs. The column will get longer since you are adding "dry load" C18 each run.

Great idea! Thank so much for your help