TLC

[aniket]

hi
i am trying an isolation of impurities i am utilising MP CHCl3:Ch3OH 8:2
i am getting optimum sepration on Precoated paltes
i ma trying the same on to glass plated 1mm thick
so kindly help me
regarding on this
pls
aniket

[Victor]

Aniket,

I haven't made TLC plates for years, but I could never make plates myself using a spreader as good as those that I could buy from manufacturers like Merck. However, the commercial plates can be very expensive.

A 1mm layer is quite thick-we used to use those for preparative TLC applications where the silica was scrpaed off and the separated compounds extracted. I think a standard layer thickeness for analytical work is 0.25mm-have you tried this?

[aniket]

Thank you so much for replying
i am using 1mm thick layer for preparative TLC for isoaltion of components
i have also tried on to Precoated plates from E.Merck(5 micron) i am getting a good separation between my com[ponents but when i transfer my TLc method on to the preparative layer chroamtography iam not getting a good resolution between my components?
can you please help me on this
can im do the isolation on to precoated HPTLC paltes of merck
pls suggest
also i have to make my own plates as the prepartaive layer chromatography plates are not affordable to me. so i have to make my own plates
also i am using 1mm thickness as 0.5- 2 mm thickness is used for preparative TLC.
also what other chroamtogrpahic method can i use to sepater components of my mixture which is cost effective
looking forward for your reply
thank you
this is Masters Of Pharmacy Project
aniket A naik

[tom jupille]

Many, if not most, precoated plates contain binders and/or fluorescence additives which can change the selectivity. That means that it may not be possible to exactly duplicate a separation carried on on a precoated plate on a handmade.

Another critical factor is water content. You may have to dry (or re-hydrate) your plates in order to match the activity of the pre-coats.

If this were my project, the first thing I would do is to try making an analytical-scale plate (0.25mm) and duplicating your analytical scale separation there. Note that you may well have to modify your solvents somewhat in order to compensate for differences in the silica, as noted above).

Once you have accomplished that, you can do a loading study on your own analytical scale plates to determine the sample load at which you start to lose resolution.

Then, repeat the separation on 1mm plates, quadrupling the maximum load obtained with the 0.25mm plates. You may still have some problems. Coating plates is something of an art, and my experience (many years ago) was that it was more difficult to get a homogeneous layer on a thick bed than a thin one.

Good luck!

[aniket]

Thank you for reeplying
but is it posibel to do an isolation experiment on to precoated plaes?
can i isolate sufficient so that i can perform spectroscopic studies
can you help me on thuis?
i would appreciate your help
thank you

[Victor]

Aniket,

Now I think I see the question you are asking us. There is nothing to stop you using pre-coated analytical TLC plates for preparative TLC. The capacity of the plates will be less- if the layer is 0.25mm the plate capacity will be a quarter of that of the 1mm plate. However, this may still be sufficient for your separation. If you put too much on, the quality of your separation will be reduced. Remember you can apply the sample as a band along the length of the plate for prep separations so that you are using the whole of the plate.

Tom's advice is good. Because of the binders used in pre-coated plates, it can be more difficult to scrape off the silica without it going everywhere, but it is not impossible to do this.

And finally-take care making your own plates. THe silica goes everywhere. I have heard of cases of silicosis from people who work in this area.

[aniket]

Thank you for replying victor
i just try and do my sepratio on precoated plates
but is thsi is the way it is done?
what will u do if you wnat to isoalted compounds from mixture by TLC, as you can not afoored prep HPLC method
what is your advice regarding this
also have done something like this ever in your life?
also i just want enough amt for spectroscopic studies.
i am not intrested in isolating a larger amt
pls help me
thank you
niket

[chromatographer1]

You might want to try using column or flash chromatography. This is a vertical column bed of silica. You can buy commercial editions like the one from Supelco or you can make your own. This is somewhat easier than TLC plates and you can use simple cheap items like glass pipettes and glass wool to start and to learn the basics.

This also has the possibility of adjusting your solvents composition or even adding solvents serially until you get the separation you desire. You also have an easier scale-up if needed.

It can be a powerful tool if used correctly. I have used it to separate synthetic dyes that have impurities that contaminate and ruin expensive LC columns. Hopefully you will have a means of visualizing your band of interest as it chromatographs down the column or as it exits the column into your collector. Good luck.

[Victor]

Aniket,

You may well find the advice of chromatographer 1 more suitable.

For TLC, again I am not absolutely sure what question you are asking. If you are doing prep TLC ( and you can use analytical TLC plates for this if you have to) you scrape off the silica containing your separated solute. You then need to extract the compounds from it. You can do this e.g. by ultrasonicating the silica in a strong solvent-maybe methanol will do for your compounds. THen you can try to see if you have enough for spectroscopic identification.

If you spread the sample as a band on the plate (say over 80% of the width of the plate) you can spot a conventional sample on the remaining part of the plate. You can use this spot to locate where you compounds of interest are (you can spray this bit of the plate if necessary while covering the band part with a glass plate to protect it from the spray). In this way you can locate your components of interest without contaminating them with spray.

[aniket]

Thank you victor

thak you for being so co-operative and helping me

what i am doing is using analytical TLC plates for Prep. TLc.

i have a mixture of 6 components and one of the component is prsent 70 % of total mixture, i am not intrested in that component, i want to isolate the rest 5 compounds i have tried doing on TLc plates i am afraid will i get enough for spectroscopic studies
can u help me on this
also i am not bale to afford Prep HPLC,

pls help me one more time

can i have your contact details

sorry if i am bothering you

aniket

[Nathan]

Aniket,

Have you ever tried OPLC ? It is an alternative to preTLC and maybe can help. It is best to use the 5µm particle size of analytical layers for best resolution, since the prep layers are ususally 25-30µm particle size and this kills you. OPLC is a pumped flow technique so band diffusion (longitudinal) is less of a problem, meaning again that resolution is better than in the case of prep TLC. And since you can elute your samples , instead of scraping, you can reuse the layers several times. Usually you can get loadings of up to 10mg. To go to higher loading, you need a bgood understanding of the sample and whether there are several compounds nearby that can interfer. Anyway, you might contact Chromeline.

[aniket]

thanks for your valuabel suggestion
i think that is a very good suggestion
but it is not possible to me to do OPLC for me here because of unavliability of equipment in my university
i have to try to do on prep TLC, so can u give me some suggestions on this
will pls guide regarding this
pls help me on this
tahnk you