< 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 µ is busted in the text as it changed to 'E' everywhere I had it. I edited it out as a correction)