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Author: Subject: Best way to quantitatively spot TCL plates?
FrankMartin
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[*] posted on 3-2-2011 at 20:05
Best way to quantitatively spot TCL plates?


I need a consistant way to spot TLC plates with 2 microLitres of sample. Normally I use thin glass 2uL capilliaries but these do not seem to give a consistant result. Is there any other way, such as with a "microsyringe"? The results are to determine reaction yields and so the samples must all be of the same weight/volume.

Frank
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Ozone
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[*] posted on 3-2-2011 at 22:07


I like to use 10 uL Hamilton (or SGE) syringes. Used for GC injection they are (when clean:P) very smooth, accurate (fine point) and reproducible. 1uL makes nice spots. Just be careful not to poke a hole into the plate when you spot it (this doesn't seem to cause too many problems--but, some of your stuff will *definitely* stay behind with the hole).

Most other techniques are tricky if you are interested in quant. densitometry. Most all suffer not from getting sample to the plate, but rather from getting all of the sample onto the plate. For example microcaps are 0.5uL, but I have almost never been able to completely empty one onto the plate, much less in a spot small enough to give decent resolution. Despite this:

I also like microcaps (very expensive here: http://www.voigtglobal.com/manufacturer/652-Chromatography-T...), but the cost might have you pulling El-cheapo Pasteur pipettes into capillaries. These work well too, and are super cheap, but pulling them is a pain in the ass. You will learn that hot glass looks just like cold glass.

The most important thing that I have noticed, is to make sure that your spots are *Completely* dry before running the plates. I can't stress this enough, particularly when you are spotting from a polar solvent such as methanol, water, or God help you, DMF or DMSO. I find that a heat gun works really well. Don't worry if you trash your first (20 ;)) plate, you will get it eventually.

Cheers,

O3



[Edited on 4-2-2011 by Ozone]




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FrankMartin
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[*] posted on 4-2-2011 at 14:57


Quote: Originally posted by Ozone  
I like to use 10 uL Hamilton (or SGE) syringes. Used for GC injection they are (when clean:P) very smooth, accurate (fine point) and reproducible. 1uL makes nice spots. Just be careful not to poke a hole into the plate when you spot it (this doesn't seem to cause too many problems--but, some of your stuff will *definitely* stay behind with the hole).

Most other techniques are tricky if you are interested in quant. densitometry. Most all suffer not from getting sample to the plate, but rather from getting all of the sample onto the plate. For example microcaps are 0.5uL, but I have almost never been able to completely empty one onto the plate, much less in a spot small enough to give decent resolution. Despite this:

I also like microcaps (very expensive here: http://www.voigtglobal.com/manufacturer/652-Chromatography-T...), but the cost might have you pulling El-cheapo Pasteur pipettes into capillaries. These work well too, and are super cheap, but pulling them is a pain in the ass. You will learn that hot glass looks just like cold glass.

The most important thing that I have noticed, is to make sure that your spots are *Completely* dry before running the plates. I can't stress this enough, particularly when you are spotting from a polar solvent such as methanol, water, or God help you, DMF or DMSO. I find that a heat gun works really well. Don't worry if you trash your first (20 ;)) plate, you will get it eventually.

Cheers,

O3



[Edited on 4-2-2011 by Ozone] Thank you. Very interesting. I have been using some miscellaneous microcaps (from the old days) of 25uL size. These work but as you mention, they are hard to empty completely. I have found this site http://www.discoverysciences.com/product.aspx?id=4272 which sells many types together with a bulb for forcing out the sample completely. I have also a Hamilton micro-syringe but since I have so many tests to do (16 per run) this would be tedious to use. I already have some free-ware densitometry software which I can use by photographing the developed plates with a digital camera. Can I use the micocaps twice, one sample per end?
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peach
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[*] posted on 4-2-2011 at 18:15


I have used fine syringe blades before.

You can sand the point off the blades without too much effort and then give them a rinse with some solvent. Or buy the blunt ones straight off. You can find the blunt ones on eBay for dispensing glues and such.

In terms of quantitative analysis, I would assume you have some method of collecting the spots and determining their content. If you're trying to determine yield by simply looking at the plate, you don't need to know the spot quantity, only the ratio of the product to the rest.




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Ozone
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[*] posted on 5-2-2011 at 00:16


If you are *very* careful you can use both ends. Otherwise, you can rinse the capillaries with solvent and re-use them. But, if you are going to do that, you might as well use the syringes (rinsing is a pain, either way). Which densitometry software are you using?

Cheers,

O3





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FrankMartin
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[*] posted on 5-2-2011 at 16:40


Quote: Originally posted by Ozone  
If you are *very* careful you can use both ends. Otherwise, you can rinse the capillaries with solvent and re-use them. But, if you are going to do that, you might as well use the syringes (rinsing is a pain, either way). Which densitometry software are you using?

Cheers,

O3

I have found freeware at "CP Atlas 2.0" (type this into Google), and also there is a good Russian one (albeit expensive, at http://www.sorbfil.com
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peach
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[*] posted on 5-2-2011 at 18:33


Nice find Martin!



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[*] posted on 5-2-2011 at 23:13


it is one like TLSee
http://www.alfatechspa.com/index.php?lang=en&mod=PRODUCT...
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peach
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[*] posted on 5-2-2011 at 23:24


I see Camag have a thing that looks like a tile cutter for doing plates, which is something I was wondering about a while back.

This scanner looks nice, it's probably outside my £9.99 budget.



Quote:
The new CAMAG TLC Scanner 4 is the successor of TLC Scanner 3 and is the most advanced workstation for densitometric evaluation of Thin-Layer Chromatograms currently available. It is based on time proven technical principles known from the previous series.

Important improvements were made in design, especially related to handling and electronics.

Analysts can now benefit from a wider spectral range for specific applications and better reproducibility thanks to improved signal to noise ratio and an optimized scanning/measurement stage.

Some features:

* Measurement of reflection, either in absorbance or fluorescence mode
* Object formats up to 200 x 200 mm
* Extended spectral range 190 – 900 nm (previous Scanner3 190 – 800 nm)
* Optimized scanning/measurement stage for even better reproducibility
* Improved signal to noise ratio
* Data step resolution 25 – 200 μm
* Fully automatic scanning; speed 1 – 100 mm/s
* Reproducible data within minutes
* Up to 36 tracks with up to 100 substances per track
* Operated with easy to use winCATS software
* winCATS options for TLC Scanner:
o Quantitative Evaluation incl. Subcomponent
o evaluation,
o Dual- and Multi-wavelength scan,
o Scanner Self Testing,
o Track Optimization,
o Spectrum Library
o Complies with GMP/GLP and 21CFR11


[Edited on 6-2-2011 by peach]




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