Sorry, but I'm a little confused. What solvent system was recommended for column chromatography?

Also, keep in mind that the best solvent system for resolving peaks on TLC may not be the best for resolving compounds on column chromatography. Spots in TLC are measured by Rf (retention factor). A spot with an Rf of 0.8 is well resolved from one with an Rf of 0.7. In column chromatography, peaks are measured by column volumes (CV). The conversion of to CV is CV=1/Rf. So a spot with an Rf of 0.8 elutes in 1.25 CV; a compound with an Rf of 0.7 elutes in 1.43 CV, a difference of 0.18 CV. If the solvent were weaker, (causes the spot to run slower up the TLC and slower in the column), you will have improved resolution. All of this assumes the TLC is the same material as the column (such as silica). For some examples of converting Rf to CV, please see:

http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Diol_Columns-Pretend_They're_Normal_Phase.pdf

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

You can also get a book that describes column chromatography from http://www.isco.com/flashguide/ (full disclosure- I work for Teledyne Isco) or from your Teledyne dealer Analisa ( WWW.ANALISA-SCIENTIFIC.COM )

Sorry, but I'm a little confused. What solvent system was recommended for column chromatography?

Also, keep in mind that the best solvent system for resolving peaks on TLC may not be the best for resolving compounds on column chromatography. Spots in TLC are measured by Rf (retention factor). A spot with an Rf of 0.8 is well resolved from one with an Rf of 0.7. In column chromatography, peaks are measured by column volumes (CV). The conversion of to CV is CV=1/Rf. So a spot with an Rf of 0.8 elutes in 1.25 CV; a compound with an Rf of 0.7 elutes in 1.43 CV, a difference of 0.18 CV. If the solvent were weaker, (causes the spot to run slower up the TLC and slower in the column), you will have improved resolution. All of this assumes the TLC is the same material as the column (such as silica). For some examples of converting Rf to CV, please see:

http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Diol_Columns-Pretend_They're_Normal_Phase.pdf

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

You can also get a book that describes column chromatography from http://www.isco.com/flashguide/ (full disclosure- I work for Teledyne Isco) or from your Teledyne dealer Analisa ( WWW.ANALISA-SCIENTIFIC.COM )

You should use the solvent that gave you the best resolution on TLC beacuse the Silica CC acts similarly as TLC. If your compound is very polar it may be retained in the Silica (CC), if so, better use Sephadex LH-20.

Hello, the best solvent system you got from the TLC should be used. Remember to start with less polar solvent (when using silica as stationary phase) and gradually increase the polarity of the gradient elution process.

thanks for the answers. I was trying to purify glucosinolates.The method for CC suggested to me is using deae sephadex a-25  silica (ion exchange chromatography). i was confused because the solution used for elution is K2SO4. no any other organic solvent used

For the TLC, i found out the best solvent system was acetone and hexane.

DEAE Sephadex A-20 isn't a silica based column, it is a weak anion exchange column based on cross-linked dextran. The mechanism for this column is very different than for silica TLC or silica column chromatography. The sulfate ions in the K2SO4 displace your glucosinolates and cause them to elute down the column.

As for the silica TLC results, I suggest running a small column initially. I test elution by running a small column to verify the compound elutes unless I have some reference compound for comparison that I can run on the TLC and verify the reference runs the same way. The solvent mixture that makes a good TLC plate is usually too strong for good column chromatography, so I would consider a gradient starting with less acetone than in your TLC experiment and increasing the concentration of acetone as I run the column, as Dr. Bismark is suggesting.

I Think you have to use RP TLC, so we could see the separation and make sure not only in one mobile phase but use gradient mobile phase because it can be use for fractination using CC

Normal  or reversed  phases TLC are used to verify the purity of compounds. Also are used to find the solvent system which separate better the components of extracts. CC are used to isolate compounds from extracts. I think that the best system to resolve spots on TLC is not always the same for separating compounds.

Have you tried Acetonitrile: water? You can try Acetonitrile: water system (eg. 8:2 or 3:7). If you get good separation, I think you can use same or (lesser polar system) for separation using simple silica based column chromatography.

I suggest you to check the separation on RP-TLC in different ratios of water/methanol or water/acetonitrile.

then use RP-column

If you prefer to use a normal phase silica based CC, you can use a normal phase TLC to find out the best solvent system for your CC. If you use RP system for CC, you should optimize your CC system by RP-TLC. But, if you use anion or cation exchange for CC system, you can't optimize by silica based TLC system. Its depend on what kind of stationary phase you use on your CC system. Unfortunately you didn't tell what kind of the stationary phase on your CC system, its hard to discuss.

stationery phase of the cc system is deae sephadex A-25

Okay. Jack already mention couple days ago. Sephadex A-25 is completely different than silica base TLC, it is an anion exchange stationary phase. You can't adjust your CC system using silica base TLC. You can perform your cc using a gradient system of methanol-water or ethanol-water start from 50% methanol/ethanol to 100 %. In my lab I used sephadex LH-20 with ethanol or methanol only to purify bisanthraquinone (possessing more than 3 hidroxy moeity). Other way, probably you can try to use normal phase silica with a solvent system chloroform-methanol-water (6:4:1). This solvent system is widely applied to separate and purify flavonoids. You can also put some amounts of mild acid in your system if your compound is stable enough.

Replace MeOH with isopropanol. Check on TLC first.

That right. If you can't get good separation by using MeOH, please follow the Khedr's advice.

Hi,

If you are planning to use silica-based separation methods, there is prep-TLC when you have a glass plate with a thicker layer of silica. Of course, you will have to run it in a bigger chamber using the solvent system of choice. The system should be chosen to provide the cleanest separation of your target band from the impurities (Rf doesn't matter) After you locate your product on a plate (UV), you can just scrape off the silica from the plate and extract your product with methanol or other appropriate solvent. Don't use stains though.

For the column chromatography, first choose the solvent system for the TLC which gives your compound Rf ~0.2 and use this system for CC. Take the amount of silica 35-45 mL per gram of your extract (yes, by volume, measure it with graduated cylinder). Choose the column which will give you for the calculated amount of silica a layer with approximately 1:4 or 1:5 diameter to height ratio. Place your extract on top of the column either in solution or co-evaporated with small amount of silica and run. Don't forget to fractionate and check TLC of fractions. Please let me know if you need more details on running CC.

Good luck!

This works for me: I do make the solvent system to be use on CC to be less polar than the actual solvent systems that resolve the components well on TLC. For example, if on TLC 50% Ethly Acetate in hexane resolve my components well, I will then use 20% Ethyl Acetate in hexane on CC.