TLC chromatography help

Tom McCloud mccloud-tom at worldnet.att.net
Wed Aug 30 19:04:51 EST 2000


Mehrdad,     The short answer  is that you can apply your sample to
the thin layer plate either as spots or as streaks.    Either will
work, but there are a great many considerations for choosing the
method you use. 
                        If you have a large number of samples you
might choose to spot  them ~<1cm apart, along with 2 or 3 suitable
controls.   Dissolve your sample in a solvent in which it is
completely (no lumps)  soluble, and  which penetrates down into the
adsorbent layer.   You don't want your sample laying only on the top
of the layer -- and that can happen with the wrong solvent, or with a
sample that is too viscous or too concentrated.  (There are some
sample/adsorbant combinations in which penetration is a problem.  A
trick that usually solves the problem is to apply a tiny amount of
methanol to the point where you will apply your sample, and then add
the sample on top before the methanol has completely evaporated).
Also the solvent should be as volatile as possible so that it is
easily removed  and not retained on the plate ( i.e. DMSO or pyridine
are bad choices).  You want to make your application spots as small as
possible, because following development the resolution of components
cannot be better than the diameter of the spot of application.
Another bad thing to do is to make multiple applications to the spot
with a solvent that causes the spot itself to develop outward, so you
are doing circular chromatography at the point of application.    You
are creating a 'donut' with very little sample in the middle of the
spot.   So the proper solvent will be one in which the sample is only
modestly soluble, and you should make several applications of very
small volumes rather than a couple applications with a lot of volume.

       You can also apply your samples as very thin lines several mm
long, but fewer samples per plate than with spots.   The same
considerations for making good spots apply to good lines, i.e. choice
of solvents, very narrow line, etc.   One advantage of the line method
is that it is sometimes possible to detect very minor components of a
complex mixture that  would be hidden (partly covered) by the spot
method. 

      But the best thing to do is to try both techniques with your
samples and see which gives the best resolution.

      Tom McCloud    SAIC/Frederick Cancer Research

 
 

On Wed, 30 Aug 2000 08:40:18 GMT, Mehrdad
<mehrdad.motamedi at valapharma.se> wrote:

>Dear Friends
>I need help with sample application on TLC (Thin layer chromatography).
>If in the method used fro sample application is recommended to to apply
>samples as "bandwise", is that possible to apply samples "spotwise"?
>I appreciate to replay to my email address directly.
>thanx in advance
>Mehrdad






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