ClearLight (Hive Addict)
05-12-03 04:06
No 432737
      Making your own TLC Standards  Bookmark   

< Commentary and corrections invited! >

  In working w/ Thin-Layer Chromatography plates it is necessary to create a "Standard" to measure the progress of your reaction as well as to determine what's in your final mix.

   Since TLC is sensitive to about 5-25 micrograms of material, it is important NOT to overload your plate.  Having a standard you can compare your reaction product to allows you to have some semi-quantitative idea of just how much you got...

  To create your standard, use the following procedure.

  1.  Take 5mg of your known product (hopefully purified) and add to 5ml of suitable solvent. Methanol is often used but other solvents may be used.

  2. Purchase a set of 10 microliter spotting pipettes for delivering an accurate amount of material.

     The as the following equation indicates, this should deliver 50 micrograms of standard to the plate

    5*E-3 gms/5e-3 liters  *  10e-6 (10micro liters) = 5e-8 gms of material (50 micrograms)

    A 5 microliter spotting pipette (also known as 5 lamda) will deliver 25 micrograms of material to the plate.


 Use w/ Sample

  Standard solutions: Prepare a 100% (5 mg of material in 5 ml solvent) and a 80% (4 mg of material in 5 ml solvent) as your standard solutions

   Prepare a 5mg/5ml solution of your sample material

   Spot the plate in positions 1 and 3 w/ the 100% and 80% Standards respectively
 
   (bee careful when spotting you want 1mm dia spots on the plate which may require multiple applications w/ the pipette, allowing to spot to dry in-between)

   Spot the plate in position 2 w/ the sample.

   If the relative spot size/intensity is between the 100% and 80% Standards, then you have a good sample size. If it is larger than the standard
   - Use less sample in the preparation 
 or
   - Use a 5, 2, or .1 microLiter spotting pipette to adjust to the 80-100% standard range.

  If the sample spot size/intensity is less than the 80% range, you can either double spot or increase the amount of sample compound in the solvent.

  Since most amines (and other drugs) quench u.v. fluorescence on the plate this method will be adequate for such compounds.

  If you are using visualization reagents, such as the tryptamine modified salwoski-van urk reagent, or Marquis reagent, check the document on rhodiums site or references to determine the sensitivity range of the reagent to your target material.  Most reageants have a sensitivity of about 5micrograms of material, although some are only good down to 25 micrograms target compound.

( It appears the micron symbol is busted in the text as it changed to 'E' everywhere I had it. I edited it out as a correction)

Infinite Radiant Light - THKRA
 
 
 
 
    bones
(Stranger)
05-12-03 06:18
No 432759
      nice info ... solvent polarity is reallllly...  Bookmark   

nice info ... solvent polarity is reallllly important to get seperation... silica is very polar... so polar substances will stick to it more readily than non polar things...ie they'll move more slowly... it is often necessary to trial several solvents of varying polarity to ensure that the sample moves slow enough to get seperation..to do this... mix some pure starting material with some pure product in roughly a 1:1 ratio ... then run them on the plate and adjust your solvents so that you can seperate em... very often mixed solvents are used... say a 3:1 mix of cylcohexane and DCM ...or whatever ... you can usually find someone whos done the solvent work for you :D ...

the point about not overloading the matrix is also very relivent :D .. this inhibits seperation.

you can make silica plates... if you can get ahold of silica... they are expensive to buy.. .about $150 ish per box where i am ...but you can cut em fine so a box should last ages!

for colourless compounds you'll need uv light :( ... but you can possibly use one of those black tubes they use at nightclubs ... but i dont know
 
 
 
 
    ClearLight
(Hive Addict)
05-12-03 10:41
No 432794
      UV light..  Bookmark   

Uv lights to use are shortwave lamps for mineral specimens.. you can buy them for $35.00 or so.. long wave blacklights from head shops are around 400nm and won't work on the plate. Make sure your uv lamp has a uv filter.. certain auction sites sell a mecury vapor light that is useless w/ out a filter

 prices range for 100  5x7 cm plates from $279.00 to $134.00  A kilo of roll you own gel (silica G 60 f245 ) is about $60-$120.

  To move polar amines etc.. oftentimes 1 - 2.5% NH4OH is used to give a better separation.  Also some folks pre-dip the plates in NaOH solution and dry for same reason.

  I saw a cute trick where they turned the 5x7 on it's side and developed it across the short axis... poor man's 25x25 plate! Excellent for spot check of column fractions..

Infinite Radiant Light - THKRA
 
 
 
 
    Osmium
(Stoni's sexual toy)
05-12-03 11:01
No 432795
      Don't go overboard with the ...  Bookmark   

Don't go overboard with the dilution/concentration game.
For reaction progress control the 'one drop plus some solvent squirted on top' dilution technique, or even 'take some of the reaction solution and put it on the plate' approach will usually be good enough.
25g is awfully little, and many impurities might evade your detection attempts when present in such low concentrations. Do not operate too close to the detection limits.

I'm not fat just horizontally disproportionate.
http://www.whatreallyhappened.com
 
 
 
 
    Lilienthal
(Moderator)
05-12-03 22:42
No 432842
      You can also put a vial with concentrated...  Bookmark   

You can also put a vial with concentrated ammonia into the chamber to basify everything. That way you don't have to put water (from ammonia) into the eluent.
 
 
 
 
    ClearLight
(Hive Addict)
05-12-03 23:11
No 432850
      THANKS!  Bookmark   

Wow lil, that is brillant!  thanks for the tip!

Infinite Radiant Light - THKRA