Chromatography Forum

Hi, I'm Felicia, a newbie.
I'm trying to separate 2 substances in my TLC plat - chloramphenicol and lidocaine HCl
trying a lot of mobile phase till Iknow we have to add a base to the mobile phase in order to show the lidocaine spot clearly in 254 nm. since it's E 1% 1cm relative low in comparison to chloramphenicol.

However, I still find another challenge to be solved ASAP, after developed the plat with mobile phase, there are always 'A black line across my developed TLC plat'. Unfortunately, it was located near the lidocaine spot so the densitogram showed there's 2 peaks with no resolution.
I have tried : n heksane : chloroform : dietilamine
n heksane : chloroform : acetone : dietilamine
toluen : aceton : dietilamine


Please help me! Thank you for your comment and any suggestions that I really appreciate that!
Have a nice day!

When you run TLC using mixed solvents, "demixing" occurs because the more polar solvent is retarded somewhat by the adsorbent. The result is a secondary solvent front (in the case of ternary or quaternary mixtures, you can sometimes see 2 or 3 secondary fronts. In addition, if the plate was not washed before using (or was stored for a long time after washing), they can adsorb contaminants which are washed forward by the secondary front to generate the "line" that you saw.

Demixing can be minimized by equilibrating the plate with vapor from your mobile phase before running (suspend the plate in the tank but don't actually put it in contact with the liquid solvent).

The above comments apply to silica gel (normal phase) plates.

Hi

Yes as Tom suggested equibration may help, but also preeluting plate with mobile phase, letting it dry (not too hot) before application can help. Cooling plate in exciator with drying agent may also be helpful.

If I find it tomorrow, I might have something related (prilocaine by TLC) to share.

Okay, thank you for your reply Mr. Tom Jupile and Mr. Krickos. I'll try it next Saturday. Really hope for significant changes. See you then in the next good news. =D
@Mr krickos: thank you, if you find the file of prilocaine to share, I appreciate it alot. You may send to my email ph_felicia@yahoo.com. Thank you! Have a nice day!

Hi

well I managed to dig something quite similar to what I was looking for, see below. It is a identity test by TLC for prilocaine base and should work for the same purposes with prilocaine HCl. If you increase concentration you may have to add 0,2ml or more water to Prilocaine HCl before you add ethanol in order to get it into solution.

I also found some older notes regarding using the below set up for impurity testing, with considerble higher sample concentration and TLC scanner att 200-210nm. In that case it is cruicial that you preelute the plate completly before analysis according to notes, drying should be performed in fume cupboard, no heating as it contains ether. Also when mixing mobbilephase, use a funnel, you will get a small water phase that should be discarded.

Not mentioned below, but typically you put filter paper on the side of the TLC tank, and let the tank/filterpappers saturate for 30mins.

Again, the procedure involves ether so take appropiate saftey actions if you try it.

Examine by thin-layer chromatography (2.2.27), using a TLC silica gel GF254 plate R.

Test solution

Dissolve 20.0 mg of the substance to be examined in alcohol R and dilute to 5 ml with the same solvent.

Reference solution (a)

Dissolve 20.0 mg of prilocaine CRS in alcohol R and dilute to 5 ml with the same solvent.

Reference solution (b)

Dissolve 20.0 mg of prilocaine CRS and 20.0 mg of lidocaine CRS in alcohol R and dilute to 5 ml with the same solvent.

Apply to the plate 10 ul of each solution. Develop over a path of 12 cm, using a mixture of 1 volume of concentrated ammonia R, 5 volumes of methanol R and 100 volumes of ether R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots.

Thank you Mr. Krickos.=D I'll try it again since the experiment last Saturday didn't give a good result yet. still there was a black line across the plat. =(

Dear guys,
i think there is a possibility of small cracks in plate or in the silica application chamber.
There is also a possibility of analyst error on application, can you change the analyst and try the same?
Why can't you try for ready made plates available in market?

I think so Felicia you're working with some ointment and its component excipients ointment base.
In this case, I recommend the show plate modified reagent Dragendorfa.
Ideally, these undivided fraction scraped off and eluted with chloroform with ammonia. And on the HPLC-MS. To know what it was. However, I think that you have to LC-MS there is no access.
PS
From cleaning TLC plate ( regard Tom Jupille "said for this") - put plate in ethalole , going ethanole at the finish and dry (activate) plate (105-120 C 2h).Thus I was able to clean up even the Czech Silufol, lying in a chemical laboratory probably since the Brezhnev era.

like said before a balck line all over the plate means that it is a "system wide problem"

it can come from the plate. wash it as said earlier, but do it with good grade solvents.
it can come from the solvents. what grade are they? if they are old or of low grade then the impurity from them can be the reason for the black line
it sometimes come as well from the tank
how are you sealing your lid on the tank?
do you use some silicon or other "gluying" material for tight seal?
this can over time go down into the chamber and make problems to chromatography

Thanks to all of your comments.

As I tried more than ten times, I become more and more realized and assured that the problem come from the interaction of the combination in mobile phases. Why? because when I'm trying to separate my substances with toluen : n hexane : diethylamine, the plat was okay! Clear! No black line. However, I can't use that mobile phase since the chloramphenicol can't move from the starting point as we know, no interaction with the mobile phase.

Actually this black line may occur in other developments as well, it's just so usual as I can say that. however it becomes a problem when my substances located near the black line. It's kinda black line under the end of development, width approximately 1 cm, greyish.

Help...

Run some blanks with suspected mobile phase that's causing the problem. Process of elimination.

cody is right, run blank with the same mobile phase.

I have already done it before, many times.. however the peak was in different shape. I suspect the peak on the developed TLC is related to the lidocaine HCl, the interaction with mobile phase because when I try to develop it with chloramphenicol, lidocaine Hcl by methanol only, the peak was appeared. It seems undefined, I know. Do you know what is the characteristic of lidocaine Hcl that make it so hard to be analyzed. I searched many books, journal, though it seems Lidocaine Hcl is just okay, no problem in developing it...

Hi

Well thing is, anything in lidocaine or chloramphenicol would stay in the moving direction unless you have submerged the application spots in the mobile phase which you are not supposed to do. It wont move backwards and react with mobilepashe covering the lower end of the plate.

Consequently it should be mobilephase related or plate cleaniess related. The issue describe is unknown to me and my collegues and we analyzed lidocaine and prilocaine alot in the past with TLC.

As you state it appears in other developments as well it surely seem non sample related.

Dietylamine might be the issue, it is usually not so pure and usually more reative than triethylamine or ammonia. Could you please confirm if you have:
a: tried preeluting plates and
b: the mobile phase I suggested earlier?

Hi, everybody! Morning!
Do you all have any idea why nhexane, toluene, diethylamine are useful in lidocaine HCl and chloramphenicol separation? I mean when we do not add diethylamine the lidocaine spot can not be detected by UV denstometer. Thank you.