[emmalaa]

I am following a thin layer chromatography protocol where the solvent system is CHCl₃/MeOH/NH₄OH mixture (65:25:4, v/v).  At hand I only have a 35% solution of NH4OH.  Is the system calling for a ratio of 4 of the 35% mixture?  And, are these ratios important?  What is the role of the NH4OH?
Many Thanks!

[Doc Oc]

Well, the main problem is that ratio doesn't add up to 100%, so whoever wrote it obviously made a typo or doesn't know what they're doing.

Can you tell us what it is you're trying to separate?  In general I try to avoid the chloroform/MeOH combos unless it's something like a peptide that just refuses to move in hexane/EtOAc.

[moov]

well Bone,i need to share one thing the purpose of ammonium hydroxide is to avoid the spreading of your spots in TLC.And i hope so ratio is important,if it is too high too bad in your elution patern.ya better to tell us what type of compounds you need to separate ?

[AC Prabakar]

Genarally for the separation of highly polar components this kind of polar mobile phase will be used.In some cases to make the spots' shape good ammonium hydroxide will be used(especially when basic(amine)functionality is there).
And unless mentioned the ratio of NH4OH we can take as 35% as it is available commercially 35% solution.
After checking with 35% if the desired separation not achieved then you can go for addition of excess of 35% soln. or increasing the strength of NH4OH.

[OC pro]

It doesn´t have to sum up to 100%. 65 parts of CHCl3, 25 parts of methanol, 4 parts of NH4OH, that´s it. Something like ~3,6:1 ratio. Quite polar. Silica gel can be dissolved from the column. I would not use more polar than 4:1 ratio CHCl3/MeOH.

NH4OH is buffering the solution so that e.g. amines will move better on TLC. Silica gel has a slightly acidic surface. In general, 1% of NH4OH is enough (means 10 ml in 1 liter of eluent). And of course, it´s an aqueous solution. But it´s only 1% and the water will be removed azeotropically on the rotavapor.  

Another typical additive is acetic acid most commonly used when trying to separate/purify acids.

[Doc Oc]

Thanks for the info, that's actually very useful considering I need to purify peptides on occasion and I hate running flash columns on them because they streak so bad.

OC pro, I didn't think of it like that, you're right.  I just make all of my stock solutions to 100% so when I saw it get to 94% I figured it was a typo or something.

[ardbeg]

If NH4OH doesnt help the streaking of basic compounds you can also try 1% triethylamine instead.  Or use basic alumina as the stationary phase.